Fl ELD OF THE I NVENTI ON

The present invention is broadly in the field of cell engineering, more precisely in the field of delivering a cargo such as a nucleic acid or a protein into cells.

BACKGROUND

The delivery of various macromolecules into the cytosol of different cell types is required for a variety of applications such as cancer imm unotherapy, stem cell therapy and other biomedical research areas. The majority of the macromolecules cannot spontaneously cross the cell membrane as it is non-permeable for many compounds. Therefore, different techniques are needed to allow the cytosolic delivery of such cell-impermeable compounds. For several years, electroporation has been used as a standard delivery tool. Electroporation has the ability to form pores into the cell membrane following the application of electrical pulses. The cell- impermeable compounds can then m igrate through these pores from the surrounding medium into the cell’s interior. Despite that electroporation can reach high delivery efficiencies for a variety of cell types, several lim itations have been reported, including high cytotoxicity, the induction of genom ic alterations, the induction of phenotypic changes and a low delivery efficiency in several primary (im mune) cells. Hence, novel techniques, which do not affect the genotype or phenotype of the cells and which can achieve a high delivery efficiency with a high viability, are required.

For example, safe and efficient production of chimeric antigen receptor (CAR)-T cells is of crucial importance for cell-based cancer im munotherapy. While, historically, viral vectors are preferentially used to transduce T cells, they are associated with safety concerns and offer limited flexibility in terms of cargo type and size. Physical transfection methods, therefore, are gaining in importance, being readily compatible with different cell types and a broad variety of cargo molecules. I n particular, nanoparticle-sensitized photoporation using nanoparticles composed of metals, metal oxides or carbon allotropes has been introduced in recent years as a gentle method to transiently permeabilize cells, allowing subsequent entry of external cargo molecules into the cells. Unfortunately, these nanoparticles do not - or only very slowly - degrade over time, thus hampering clinical translation. I ndeed, more stringent regulatory issues apply to the medical use of non-degradative nanomaterials. I n addition, there is a generic safety concern about the use of inorganic nanoparticles, such as gold nanoparticles. Of particular concern is that fact that after laser treatment, gold nanoparticles for photoporation, for instance with an initial size of 60 nm , tend to fragment into very small nanoparticles (< 10 nm) which could potentially go through the nuclear membrane and intercalate with the DNA, causing genotoxic effects.

Harizaj Aranit et. al. (DOI : 10.1002/adfm .202102472) discloses a method for the delivery of a cargo such as m RNA into cells using relatively large photoresponsive organic particles. More specifically, a delivery of a cargo is accomplished via successful vapour bubble formation from photoresponsive organic particles upon irradiation with electromagnetic radiation. However, as that was an exceptional proof of concept work, the method is still far from to be used in therapeutics setting.

Thus, there remains a need in the art for further optimization of methods for delivering cargo molecules into cells more efficiently and with minimal impact to cell viability, especially for T cells, to bring it to the clinical use.

SUMMARY OF THE I NVENTI ON

By performing experiments, the present inventors optim ized the size of photoresponsive organic nanoparticles to obtain the most efficient delivery of a cargo such as m RNA into cells upon irradiation with electromagnetic radiation, wherein said photoresponsive organic nanoparticles is polydopamine particle.

Accordingly, a first aspect of the invention relates to an in vitro or ex vivo method for delivering a cargo into a cell, the method comprising: contacting a cell with one or more photoresponsive organic particle and a cargo, wherein the cargo is not bound to the one or more photoresponsive organic particles, and where in the organic particle has a diameter between 100 nm to 550 nm , wherein said organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a mixture of the cell, the cargo, and the one or more photoresponsive organic particles; and irradiating the m ixture of the cell, the cargo, and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell; or contacting a cell with one or more photoresponsive organic particles, where in the organic particle has a diameter between 100 nm to 550 nm , wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a m ixture of the cell and the one or more photoresponsive organic particles; irradiating the m ixture of the cell and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell; and contacting the mixture of the cell and the one or more photoresponsive organic particles with a cargo, thereby delivering the cargo into the cell.

By experimenting with the photoresponsive organic particle size, the present inventors increased the transfection efficiency, while resulting in low cytotoxicity and high cell viability, and hence high yield of delivery. The present methods open up the possibility to allow for the production of engineered therapeutic cell products, such as CAR-T cells by improving the cargo delivery into the target cell via a clinically approved precursor such as polydopam ine.

DESCRI PTI ON OF Fl GURES

Figure 1 shows the physicochemical characterization of 100 nm uncoated (black striped line) and BSA-coated PD NPs (gray lines) . Graph and table showing the hydrodynamic diameter and zeta-potential of uncoated PD NPs (black striped line) and PD-BSA NPs (gray striped dots and striped line) as measured in Hyclone water or OptiMEM by DLS.

Figure 2 shows the physicochemical characterization of 250 nm uncoated (black striped line) and BSA-coated PD NPs (gray lines) . Graph and table showing the hydrodynamic diameter and zeta-potential of uncoated PD NPs (black striped line) and PD-BSA NPs (gray striped dots and striped line) as measured in Hyclone water or OptiMEM by DLS.

Figure 3 shows the physicochemical characterization of 500 nm uncoated (black striped line) and BSA-coated PD NPs (gray lines) . Graph and table showing the hydrodynamic diameter and zeta-potential of uncoated PD NPs (black striped line) and PD-BSA NPs (gray striped dots and striped line) as measured in Hyclone water or OptiMEM by DLS.

Figure 4 shows the NP size for FITC dextran 500 kDa delivery. The delivery efficiency of FD500 in unstim ulated T cells can be increased from 70% to > 90% (Fig 4. A, C and E) . The delivery yield of FD500 can be increased from - 40% to - 60% (Fig. 4 B, D and F)

Figu e 5 shows the NP size for m RNA transfections.

DETAI LED DESCRI PTI ON OF THE I NVENTI ON

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of” and “consisting essentially of”.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of + /-10% or less, preferably +/-5% or less, more preferably + /-1 % or less, and still more preferably + / - 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as com monly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention. Unless indicated otherwise, the references to “use” or “uses” as taught herein include a medical use or medical method.

By extensive experiment testing, the present inventors have found that photoresponsive organic particles selected from the group consisting of a polymer- based particle, a protein-based particle, a lipid-based particle (e.g. a liposome or a solid lipid particle) , and a combination thereof allow to deliver efficiently and safely a cargo into cells, for instance m RNA into human T cells. Furthermore, the present inventors have optim ized the photoresponsive organic particle sizes that is most efficient for specific types of macromolecules, such as polysaccharide or RNA.

Accordingly, a first aspect of the invention relates to an in vitro or ex vivo method for delivering a cargo into a cell, the method comprising: contacting a cell with one or more photoresponsive organic particles, wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a m ixture of the cell and the one or more photoresponsive organic particles; irradiating the mixture of the cell and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell; and contacting the m ixture of the cell and the one or more photoresponsive organic particles with a cargo, wherein the cargo is not bound to the one or more photoresponsive organic particles, thereby delivering the cargo into the cell; or contacting a cell with one or more photoresponsive organic particles and a cargo, wherein the cargo is not bound to the one or more photoresponsive organic particles, and wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a mixture of the cell, the cargo, and the one or more photoresponsive organic particles; and irradiating the mixture of the cell, the cargo, and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell; wherein said one or more photoresponsive organic particles have a particle size of between 100 nm and 550 nm .

I n a further embodiment, said particle size of the photoresponsive organic particles is between 150 and 550 nm , more preferably between 100 and 500 nm , more preferably between 200 and 400 nm , more preferably 200 and 300 nm, more preferably of between 225 and 275 nm , more preferably between 230 and 260 nm , more preferably between 240 and 255 nm , such as 250 nm .

I n another embodiment, said particle size of the photoresponsive organic particles is between 150 and 550 nm , more preferably between 200 and 550 nm , more preferably between 300 and 550 nm , more preferably 400 and 550 nm , more preferably of between 450 and 550 nm , such as 500 nm .

I n preferred embodiments, said particle size of the photoresponsive organic particles is between 200nm and 300nm .

I n an embodiment, the particle size can be amended based on cell type and type of cargo molecule. For instance, when transfecting (naive) T cells with m RNA, a preferred particle size m ight be in the range of 200 to 300 nm , such as 250 nm . For macrophages, the preferred particle size might be between 400 and 550 nm , such as 500 nm .

I n the context of the current specification, the term ‘particle size’ is to be understood as the distance of the two most distant points on said particle or the largest possible diameter of said particle. For a spherical particle, said particle size will be equal to the diameter of said sphere.

Said particle size can be determ ined by means known in the art, such as Transm ission Electron Microscopy (TEM) , Scanning Electron Microscopy (SEM) , Dynam ic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) or atomic force microscopy (AFM) . I n a preferred embodiment, said particle size is determined by using Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) .

As indicated above, the methods as taught herein may be in vitro or ex vivo methods. The term “in vitro” as used herein is to denote outside, or external to, animal or human body. The term “in vitro” as used herein should be understood to include “ex vivo”. The term “ex vivo” typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g. in a culture vessel.

Alternatively, the methods as taught herein may be in vivo methods. The term “in vivo” as used herein is to denote inside, or internal to, animal or human body. The recitation “delivering a cargo into a cell” as used herein refers to bringing (or providing or introducing) a cargo into a cell.

I n embodiments, the cargo as taught herein is not bound to the one or more

5 photoresponsive organic particles. I n other words, in embodiments, the cargo and the one or more photoresponsive organic particles are not bound to each other. Hence, in embodiments of the methods as taught herein, the cargo and the one or more photoresponsive organic particles are added separately to the mixture of the cell, the cargo, and the one or more photoresponsive organic particles.

The terms “not bound” or “unbound” may be used interchangeably herein and denotes that a first component is not combined with or chemically bonded to a second component. For instance, the first component is not bound to the second component by a covalent binding or by a non-covalent interaction such as an5 electrostatic or hydrophobic interaction. The phrase “the cargo is not bound to the one or more photoresponsive organic particles” denotes that the cargo is not combined with or chem ically bonded to the one or more photoresponsive organic particles, e.g. the cargo is not coupled (e.g. grafted) to or enclosed (e.g. encapsulated) in the one or more photoresponsive organic particles. For instance,0 the cargo is not bound to the one or more photoresponsive organic particles by a covalent binding or by a non-covalent interaction such as an electrostatic or hydrophobic interaction.

I n embodiments, the cargo as taught herein is not covalently bound to the one or5 more photoresponsive organic particles. I n other words, in embodiments, the cargo and the one or more photoresponsive organic particles are not covalently bound to each other.

As used herein, the terms “cargo” or “agent” broadly refer to any chemical (e.g.,0 inorganic or organic) , biochemical or biological substance, molecule or macromolecule (e.g., biological macromolecule) , particle (e.g. a nanoparticle) , a combination or mixture thereof, a sample of undeterm ined composition, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Preferred “cargos” or “agents” include nucleic acids, oligonucleotides,5 ribozymes, proteins, polypeptides, peptides, peptidomimetics, peptide nucleic acids, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, lipids, carbohydrates, polysaccharides, etc. , and any combinations thereof such as gene editing system , e.g. CRI SPR/Cas. Depending on the context, the term “agent” may denote a “therapeutic agent” or “drug”, useful for or used in the treatment, cure, prevention, or diagnosis of a disease. The cargo as taught herein includes but is not lim ited to a cargo in solution, and a dried or lyophilized cargo, such as a powder of the cargo.

I n embodiments, the cargo may comprise or consist of two or more agents combined with or chem ically bonded to each other. I n embodiments, the cargo may comprise or consist of two or more agents conj ugated (e.g. covalently bound) to each other. For instance, the cargo may be an agent comprising or consisting of two, three, four, five, six or more agents conj ugated (e.g. covalently bound) to each other.

I n embodiments, the cargo as taught herein may be bound to a particle such as a nanoparticle (which is not the photoresponsive organic particle) . I n embodiments, the cargo may be combined with or chemically bonded to a particle such as a nanoparticle, e.g. the cargo may be coupled (e.g. grafted) to or enclosed (e.g. encapsulated) in a particle such as a nanoparticle.

The term “nucleic acid” as used herein typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit com monly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A) , guanine (G) , cytosine (C) , thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochem ically modified (e.g., methylated) , non-natural or derivatised bases. Exemplary modified nucleobases include without lim itation 5- substituted pyrimidines, 6-azapyrim idines and N-2, N-6 and 0-6 substituted purines, including 2-am inopropyladenine, 5- propynyluracil and 5-propynylcytosine. I n particular, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose com mon in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2'-0- alkylated, e.g., 2'-0-methylated or 2'-0-ethylated sugars such as ribose; 2'-0- alkyloxyalkylated, e.g. , 2’-O-methoxyethylated sugars such as ribose; or 2'-O,4'-C- alkylene-linked, e.g., 2'-O,4'-C-methylene-linked or 2'-O,4'-C-ethylene-linked sugars such as ribose; 2’-fluoro-arabinose, etc.) . Nucleic acid molecules comprising at least one ribonucleoside unit may be typically referred to as ribonucleic acids or RNA. Such ribonucleoside unit(s) comprise a 2'-OH moiety, wherein -H may be substituted as known in the art for ribonucleosides (e.g. , by a methyl, ethyl, alkyl, or alkyloxyalkyl) . Preferably, ribonucleic acids or RNA may be composed primarily of ribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be ribonucleoside units. Nucleic acid molecules comprising at least one deoxyribonucleoside unit may be typically referred to as deoxyribonucleic acids or DNA. Such deoxyribonucleoside unit(s) comprise 2'-H. Preferably, deoxyribonucleic acids or DNA may be composed primarily of deoxyribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units. Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages com mon in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3’-thioacetal, and sulfone internucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate- based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term “nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid m imetics, including, without lim itation, peptide nucleic acids (PNA) , peptide nucleic acids with phosphate groups (PHONA) , locked nucleic acids (LNA) , morpholino phosphorodiam idate-backbone nucleic acids (PMO) , cyclohexene nucleic acids (CeNA) , tricyclo-DNA (tcDNA) , and nucleic acids having backbone sections with alkyl linkers or am ino linkers (see, e.g. , Kurreck 2003 (Eur J Biochem 270: 1628-1644)) . “Alkyl” as used herein particularly encompasses lower hydrocarbon moieties, e.g. , C1 -C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2- propenyl, and isopropyl. Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof. The term “nucleic acid” preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-m RNA, m RNA, cDNA, genom ic DNA (gDNA) , plasm id DNA (pDNA) , amplification products, oligonucleotides, and synthetic (e.g. , chemically synthesised) DNA, RNA or DNA/RNA hybrids. RNA is inclusive of RNAi (inhibitory RNA) , dsRNA (double stranded RNA) , siRNA (small interfering RNA) , m RNA (messenger RNA) , m iRNA (m icro-RNA) , tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid) , and cRNA (complementary RNA) . A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chem ically or biochem ically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. I n addition, nucleic acid can be circular or linear.

The term “oligonucleotide” as used throughout this specification refers to a nucleic acid (including nucleic acid analogues and mimetics) oligomer or polymer as defined herein. Preferably, an oligonucleotide, such as more particularly an antisense oligonucleotide, is (substantially) single-stranded. Oligonucleotides as intended herein may have a length of about 10 to about 100 nucleoside units (i.e. , nucleotides or nucleotide analogues) , preferably about 15 to about 50, more preferably about 20 to about 40, also preferably about 20 to about 30 nucleoside units (i.e., nucleotides or nucleotide analogues) . Oligonucleotides as intended herein may comprise one or more or all non-naturally occurring heterocyclic bases and/or one or more or all non-naturally occurring sugar groups and/or one or more or all non- naturally occurring inter-nucleoside linkages, the inclusion of which may improve properties such as, for example, increased stability in the presence of nucleases and increased hybridization affinity, increased tolerance for m ismatches, etc.

Nucleic acid binding agents, such as oligonucleotide binding agents, are typically at least partly antisense to a target nucleic acid of interest. The term “antisense” generally refers to an agent (e.g., an oligonucleotide) configured to specifically anneal with (hybridize to) a given sequence in a target nucleic acid, such as for example in a target DNA, hnRNA, pre-m RNA or m RNA, and typically comprises, consist essentially of or consist of a nucleic acid sequence that is complementary or substantially complementary to said target nucleic acid sequence. Antisense agents suitable for use herein, such as hybridization probes or amplification or sequencing primers and primer pairs) may typically be capable of annealing with (hybridizing to) the respective target nucleic acid sequences at high stringency conditions, and capable of hybridizing specifically to the target under physiological conditions. The terms “complementary” or “complementarity” as used throughout this specification with reference to nucleic acids, refer to the normal binding of single-stranded nucleic acids under perm issive salt (ionic strength) and temperature conditions by base pairing, preferably Watson-Crick base pairing. By means of example, complementary Watson-Crick base pairing occurs between the bases A and T, A and U or G and C. For example, the sequence 5'-A-G-U-3' is complementary to sequence 5'-A-C-U-3'. The reference to oligonucleotides may in particular but without limitation include hybridization probes and/or amplification primers and/or sequencing primers, etc., as com monly used in nucleic acid detection technologies.

The terms “ribozyme” or “ribonucleic acid enzymes” as used herein refer to RNA molecules that have the ability to catalyse specific biochem ical reactions, for example RNA splicing in gene expression. The function of ribozymes is sim ilar to the action of protein enzymes. The most com mon activities of ribozymes are the cleavage or ligation of RNA and DNA and peptide bond formation. Within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link am ino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.

The term “protein” as used herein generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, sem i-synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s) , such as, without lim itation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-term inal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or m utants which carry amino acid sequence variations vis-a-vis a corresponding native protein, such as, e.g. , am ino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally occurring protein parts that ensue from processing of such full-length proteins.

The term “polypeptide” as used herein encompasses polymeric chains of am ino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not lim ited to any m inimum length of the polypeptide chain. The term may encompass naturally, recombinantly, sem i-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without lim itation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-term inal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or m utants which carry am ino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g. , naturally occurring polypeptide parts that ensue from processing of such full- length polypeptides.

The term “peptide” as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g. , 45 am ino acids or less, preferably 40 am ino acids or less, e.g., 35 am ino acids or less, more preferably 30 amino acids or less, e.g. , 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.

The term “peptidom imetic” refers to a non-peptide agent that is a topological analogue of a corresponding peptide. Methods of rationally designing peptidom imetics of peptides are known in the art. For example, the rational design of three peptidomimetics based on the sulphated 8-mer peptide CCK26-33, and of two peptidomimetics based on the 1 1 -mer peptide Substance P, and related peptidom imetic design principles, are described in Horwell 1995 (Trends Biotechnol 13: 132-134) .

The term “peptide nucleic acid” or “PNA” refers to an artificially synthesized polymer comprising N-(2-aminoethyl)-glycine (AEG) units linked by peptide bonds. The various purine and pyrim idine bases are linked to the backbone by a methylene bridge (-CH2-) and a carbonyl group (-(C= O)-) . PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the last (right) position.

As used herein, the term “antibody” is used in its broadest sense and generally refers to any im munologic binding agent. The term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2- , 3- or more- valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest, i.e., antigen- binding fragments) , as well as multivalent and/or m ulti-specific composites of such fragments. The term “antibody” is not only inclusive of antibodies generated by methods comprising im munisation, but also includes any polypeptide, e.g. , a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro or in vivo.

An antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody. An antibody may be a polyclonal antibody, e.g., an antiserum or im munoglobulins purified there from (e.g., affinity-purified) . An antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility. By means of example and not lim itation, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. 1975 (Nature 256: 495) , or may be made by recombinant DNA methods (e.g., as in US 4,816,567) . Monoclonal antibodies may also be isolated from phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624- 628) and Marks et al. 1991 (J Mol Biol 222: 581 -597) , for example.

Antibody binding agents may be antibody fragments. “Antibody fragments” comprise a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv and scFv fragments, single domain (sd) Fv, such as VH domains, VL domains and VHH domains; diabodies; linear antibodies; single-chain antibody molecules, in particular heavy-chain antibodies; and m ultivalent and/or m ultispecific antibodies formed from antibody fragment(s) , e.g., dibodies, tribodies, and m ultibodies. The above designations Fab, Fab’, F(ab’)2, Fv, scFv etc. are intended to have their art- established meaning.

The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g. , birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, m urine (e.g., mouse, rat, etc.) , donkey, rabbit, goat, sheep, guinea pig, camel (e.g. , Camelus bactrianus and Camelus dromaderius) , llama (e.g., Lama paccos, Lama glama or Lama vicugna) or horse.

A skilled person will understand that an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions) , insofar such alterations preserve its binding of the respective antigen. An antibody may also include one or more native or artificial modifications of its constituent am ino acid residues (e.g., glycosylation, etc.) .

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art, as are methods to produce recombinant antibodies or fragments thereof (see for example, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1988; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447; “Monoclonal Antibodies: A Manual of Techniques”, by Zola, ed., CRC Press 1987, ISBN 0849364760; “Monoclonal Antibodies: A Practical Approach”, by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: “Antibody Engineering: Methods and Protocols”, Lo, ed. , Humana Press 2004, I SBN 1588290921 ) .

I n certain embodiments, the agent may be a Nanobody. The terms “Nanobody” and “Nanobodies” are trademarks of Ablynx NV (Belgium) . The term “Nanobody” is well- known in the art and as used herein in its broadest sense encompasses an im munological binding agent obtained (1 ) by isolating the VHH domain of a heavychain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a VHH domain ; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain ; (4) by “camelization” of a VH domain from any animal species, and in particular from a mam malian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “dAb” as described in the art, or by expression of a nucleic acid encoding such a camelized dAb; (6) by using synthetic or sem i-synthetic techniques for preparing proteins, polypeptides or other am ino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. “Camelids” as used herein comprise old world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example Lama paccos, Lama glama and Lama vicugna) .

The term “antibody-like protein scaffolds” or “engineered protein scaffolds” broadly encompasses proteinaceous non-imm unoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random m utagenesis in combination with phage display or other molecular selection techniques) . Usually, such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin or the ankyrin repeat) . Such scaffolds have been extensively reviewed and include without lim itation affibodies, based on the Z-domain of staphylococcal protein A, a three-helix bundle of 58 residues providing an interface on two of its alpha-helices (Nygren) ; engineered Kunitz domains based on a small (ca. 58 residues) and robust, disulphide-crosslinked serine protease inhibitor, typically of human origin (e.g. LACI-D1 ) , which can be engineered for different protease specificities; monobodies or adnectins based on the 1 Oth extracellular domain of human fibronectin I I I (10Fn3) , which adopts an Ig- like beta-sandwich fold (94 residues) with 2-3 exposed loops, but lacks the central disulphide bridge (Koide and Koide) ; anticalins derived from the lipocalins, a diverse family of eight-stranded beta-barrel proteins (ca. 180 residues) that naturally form binding sites for small ligands by means of four structurally variable loops at the open end, which are abundant in humans, insects, and many other organisms (Skerra 2008) ; DARPins, designed ankyrin repeat domains (166 residues) , which provide a rigid interface arising from typically three repeated beta-turns (Stumpp et al.) ; avimers (m ultimerized LDLR-A module) ; and cysteine-rich knottin peptides.

The term “aptamer” refers to single-stranded or double-stranded oligo-DNA, oligo- RNA or oligo-DNA/RNA or any analogue thereof that specifically binds to a target molecule such as a peptide. Advantageously, aptamers display fairly high specificity and affinity for their targets.

The term “photoaptamer” refers to an aptamer that contains one or more photoreactive functional groups that can covalently bind to or crosslink with a target molecule.

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides. I n embodiments, the chemical substance is an organic molecule, preferably a small (organic) molecule. The term “small molecule” refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g. , proteins, peptides, nucleic acids, etc.) . Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da. The chemical substance, in particular the small molecule may be an imaging agent, such as a contrast agent.

The term “lipid” as used herein refers to a macromolecule that is soluble in a nonpolar solvent. Lipids may be divided into eight categories: fatty acids; glycerolipids; glycerophospholipids; sphingolipids; saccharolipids; polyketides; sterol lipids or sterols; and prenol lipids or prenols.

The term “carbohydrate” generally refers to a biomolecule consisting of carbon (C) , hydrogen (H) and oxygen (O) atoms. Usually, a carbohydrate has a hydrogenoxygen atom ratio of 2: 1 (as in water) and thus the empirical formula Cm (H2O)n (where m may or may not be different from n) . Carbohydrates encompass polyhydroxy aldehydes, ketones, alcohols, acids, their simple derivatives and their polymers having linkages of the acetal type. They may be classified according to their degree of polymerization, and may be divided initially into three principal groups, namely sugars, oligosaccharides and polysaccharides.

The term “polysaccharide” generally refers to a polymer or macromolecule consisting of monosaccharide units joined together by glycosidic bonds. Polysaccharides may be linear or branched.

The term “gene editing system” or “genome editing system” as used herein refers to a tool to induce one or more nucleic acid modifications, such as DNA or RNA modifications, into a specific DNA or RNA sequence within a cell. Targeted genome modification is a powerful tool for genetic manipulation of cells and organisms, including mam mals. Genome modification or gene editing, including insertion, deletion or replacement of DNA in the genome, can be carried out using a variety of known gene editing systems. Gene editing systems typically make use of an agent capable of inducing a nucleic acid modification. I n certain embodiments, the agent capable of inducing a nucleic acid modification may be a (endo)nuclease or a variant thereof having altered or modified activity. (endo)Nucleases typically comprise programmable, sequence-specific DNA- or RNA-binding modules linked to a nonspecific DNA or RNA cleavage domain. I n DNA, these nucleases create site- specific double-strand breaks at desired locations in the genome. The induced double-stranded breaks are repaired through nonhomologous end-joining or homologous recombination, resulting in targeted m utations. I n certain embodiments, said (endo)nuclease may be RNA-guided. I n certain embodiments, said (endo)nuclease can be engineered nuclease such as a Clustered Regularly I nterspaced Short Palindromic Repeats (CRISPR) associated (Cas) (endo)nuclease, such as Cas9, Cpf 1 , or C2c2, a (zinc finger nuclease (ZFN) ,a transcription factor-like effector nuclease (TALEN) , a meganuclease, or modifications thereof. Methods for using TALEN technology, Zinc Finger technology and CRISPR/Cas technology are known by the skilled person.

I n embodiments of the methods as taught herein (including the in vitro methods as taught herein and the in vivo methods or uses as taught herein) , the cargo may be selected from the group consisting of a nucleic acid, a protein, a chem ical substance, a polysaccharide, and combinations thereof, such as a gene editing system e.g. CRISPR/Cas system . Such cargos can be efficiently delivered into the cells by contacting the cells with the cargo and the photoresponsive organic particles as taught herein, and irradiating the m ixture of the cells, the cargo, and the photoresponsive organic particles with electromagnetic radiation.

I n embodiments of the methods as taught herein, the cargo may be a nucleic acid. I n embodiments of the methods as taught herein, the cargo may be m RNA or plasm id DNA. Using nucleic acids such as m RNA or plasmid DNA as a cargo advantageously allows the delivery of desired genetic constructs into cells. Using m RNA as a cargo allows transient expression of a construct into cells without the need for genom ic integration, thereby providing control over the duration of expression and avoiding any risks for unintended m utations by the genomic integration.

I n embodiments, the cargo may be a nucleic acid, such as m RNA or plasmid DNA, having a size of at least 0.5 kilobase (kb) . For example, the cargo may be a nucleic acid, such as m RNA or plasm id DNA, having a size of at least 0.6 kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 .0 kb, at least 1 .5 kb, at least 2.0 kb, or more. For example, the cargo may be a nucleic acid, such as m RNA or plasm id DNA, having a size of at least 3.0 kb, at least 4.0 kb, at least 5.0 kb, at least 6.0 kb, at least 7.0 kb, at least 8.0 kb, at least 9.0 kb, at least 10.0 kb, or more. Such (large) nucleic acids can be efficiently delivery into cells by the present methods. I n embodiments of the methods as taught herein, the cargo may be a protein. The present methods advantageously allow the delivery of desired proteins into cells.

I n embodiments, the cargo may be a negatively charged protein at physiological pH (e.g. pH of about 6 to about 8) . ( I EP) . I n embodiments, the cargo may be a neutral protein at physiological pH (e.g. pH of about 6 to about 8) . The present methods advantageously allow efficient delivery of such proteins into the cells.

I n embodiments of the methods as taught herein, the cargo may be a chemical substance. The present methods advantageously allow the delivery of desired chemical substances into cells.

I n embodiments of the methods as taught herein, the cargo may be a molecular contrast agent. For instance, the cargo may be molecular contrast agent such as a gadolinium chelate, a fluorophore, or a chromophore.

I n embodiments of the methods as taught herein, the cargo may be a polysaccharide. The present methods advantageously allow the delivery of desired polysaccharides into cells.

I n embodiments of the methods as taught herein, the cargo may be a particle such as a nanoparticle, e.g. a lum inescent or sensing nanoparticle. For instance, the cargo may be a lum inescent or sensing nanoparticle such as a superparamagnetic ironoxide nanoparticle, a plasmonic nanoparticle, e.g. gold nanoparticle, a quantum dot, an upconverting nanoparticle, a phosphorescent nanoparticle, a persistent lum inescent nanoparticle, or a carbon dot.

I n embodiments, the cargo may be a protein, a chemical substance, a polysaccharide, or combination thereof having a size of at least 200 kDa. I n embodiments, the cargo may be a protein, a polysaccharide, a chemical substance, or combination thereof having a size of at least 250 kDa, at least 300 kDa, at least 350 kDa, at least 400 kDa, at least 450 kDa, at least 450 kDa, at least 500 kDa, at least 600 kDa, at least 700 kDa, at least 800 kDa at least 900 kDa, or at least 1000 kDa. I n embodiments, the cargo may be a protein, a polysaccharide, a chemical substance, or combination thereof having a size of about 250 kDa to about 3500 kDa, about 300 kDa to about 3000 kDa, about 350 kDa to about 2500 kDa, about 400 kDa to about 2000 kDa, about 450 kDa to about 1500 kDa, or about 500 kDa to about 1000 kDa. Such (large) proteins, chem ical substances, polysaccharides, or combinations thereof can be efficiently delivery into cells by the present methods. I n embodiments of the methods as taught herein, the cargo is in solution. I n embodiments, the concentration of the cargo in the mixture may be about 0.001 to about 100 mg/ml (i.e. pg/pl). For example, the concentration of the cargo in the m ixture may be about 0.01 mg/m l to about 10 mg/ml or about 0.1 mg/ml to about 1 .0 mg/m l. The cargo is preferably provided in the mixture in an aqueous solution, such as in water or a cell culture medium .

The cargo may be comprised in a composition or form ulation such as a pharmaceutical formulation or kit of parts, as will be described further herein. The composition may comprise the cargo in a concentration ranging from about 0.005 mg/m l to 100 mg/m l, such as for example about 0.01 mg/m l to about 50 mg/m l.

The term “cell” refers to all types of biological cells, including eukaryotic cells and prokaryotic cells. As used herein, the terms “cells” and “biological cells” are interchangeably used.

I n embodiments of the methods as taught herein, the cell may be a plant cell.

I n embodiments of the methods as taught herein, the cell may be an animal cell. I n embodiments of the methods as taught herein, the cell may be a human cell. As illustrated in the example section, the methods as taught herein advantageously allow high delivery yield into animal cells, in particular into human cells, e.g. human T cells such as naive T-cells.

I n embodiments, methods as taught herein is particularly suitable for imm une cells such as cells derived from hematopoietic stem cells.

I n embodiments, the im mune cell is T cell, a lymphocyte, a macrophage, a dendritic cell, a monocyte, a NK (Natural killer) cell, a NKT cell, a B cell, a neutrophil, a granulocyte, a microglial cell, or a Langerhans cell.

I m mune cells develop from hematopoietic stem cells in the bone marrow and become different types of white blood cells. These include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, NK cells, and lymphocytes (B cells and T cells) . Subtypes of imm une cells vary in morphology and size. Several subtypes of imm une cells are small (6-9 pm diameter) relative to the size of most tissue-cultured cells (12-15 pm diameter). For example, there are two major types of lymphocyte: B lymphocytes or B cells, and T lymphocytes or T cells of which most of them are small at about 6-9pm in diameter. Other small imm une cells types include, NK cells with a diameter of 6-7 pm, langerhans cells, roughly 12 microns in diameter.

The inventors discovered that the method disclosed here is particularly advantageous for transfecting imm une cells such as T cells. The advantage is likely to be due to small particle size used in the disclosed method which leads to lesser levels of cell death and reaching a higher transfection yield of the small cell.

I n embodiments of the methods as taught herein, the cell may be a m icroorganism .

I n embodiments of the methods as taught herein, the cell may be a bacteria, a yeast, or a fungal cell.

I n embodiments of the methods as taught herein, the cell may be a synthetic cell.

Cells may be obtained from (e.g. , isolated from , derived from) a biological sample, preferably a biological sample of a human subject, e.g. , blood, bone marrow, trabecular bone, umbilical cord, placenta, foetal yolk sac, skin (derm is) , specifically foetal and adolescent skin, periosteum , dental pulp, tendon and adipose tissue. The term “biological sample” or “sample” as used herein refers to a sample obtained from a biological source, e.g., from an organism , such as a plant, an animal or human subject, cell culture, tissue sample, etc. A biological sample of a plant, an animal or human subject refers to a sample removed from a plant, an animal or human subject and comprising cells thereof. The biological sample of a plant, an animal or human subject may comprise one or more tissue types and may comprise cells of one or more tissue types. Methods of obtaining biological samples of a plant, an animal or human subject are well known in the art, e.g., tissue biopsy or drawing blood.

The biological sample can be derived from a biological origin, such as from plants, humans or non-human animals, preferably warm-blooded animals, even more preferably mam mals, such as, e.g. , non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like. The term “non-human animals” includes all vertebrates, e.g., mam mals, such as non-human primates, (particularly higher primates) , sheep, dog, rodent (e.g. mouse or rat) , guinea pig, goat, pig, cat, rabbits, cows, and non-mam mals such as chickens, amphibians, reptiles etc. Preferably, the biological sample is derived from human origin.

The biological sample can be a biological fluid or a non-fluid biological sample. It should be understood that sample preparation before or during the method as taught herein can release the cells, for instance from a non-fluid biological sample. Hence, the method as taught herein may comprise the step of releasing the cells from a non-fluid biological sample such as a cell tissue.

I n embodiments, the cell may be obtained from a biological sample of plant origin, plant tissue culture, or plant cell culture. For instance, the cell may be obtained from a plant cell suspension culture. I n embodiments, the cell may be a plant cell of a living plant.

I n embodiments, the cell may be obtained from a biological sample of animal origin, such as blood, bone marrow, trabecular bone, umbilical cord, placenta, foetal yolk sac, skin (derm is) , specifically foetal and adolescent skin, periosteum , dental pulp, tendon and adipose tissue.

I n embodiments, the cell may be obtained from whole blood. I n embodiments, the cell may be obtained from a buffy coat. After centrifugation of whole blood, three layers can be distinguished: a layer of clear fluid (i.e. the plasma) , a layer of red fluid containing red blood cells and granulocytes, and a thin layer in between, called the buffy coat. The buffy coat contains most of the white blood cells and platelets. I n embodiments, the cell may be obtained from bone marrow.

I n embodiments, the cell may be a blood cell, a stem cell, or a cell derived thereof. The terms “blood cell”, “hematopoietic cell”, “hemocyte” or “hematocyte” refer generally to a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes) , white blood cells (leukocytes) , and platelets (thrombocytes) .

The term “stem cell” refers generally to an unspecialized or relatively less specialized and proliferation-competent cell, which is capable of self-renewal, i.e., can proliferate without differentiation, and which or the progeny of which can give rise to at least one relatively more specialized cell type. The term encompasses stem cells capable of substantially unlim ited self-renewal, i.e., wherein the progeny of a stem cell or at least part thereof substantially retains the unspecialized or relatively less specialized phenotype, the differentiation potential, and the proliferation capacity of the mother stem cell, as well as stem cells which display limited selfrenewal, i.e., wherein the capacity of the progeny or part thereof for further proliferation and/or differentiation is demonstrably reduced compared to the mother cell. By means of example and not limitation, a stem cell may give rise to descendants that can differentiate along one or more lineages to produce increasingly relatively more specialized cells, wherein such descendants and/or increasingly relatively more specialized cells may themselves be stem cells as defined herein, or even to produce terminally differentiated cells, i.e., fully specialized cells, which may be post-m itotic.

I n embodiments of the methods as taught herein, the cell may be an imm une cell such as a T cell, a lymphocyte, a macrophage, a dendritic cell, a monocyte, a NK cell, a NKT cell, a B cell, a neutrophil, a granulocyte, a microglial cell, or a Langerhans cell. I n embodiments, the imm une cell is a lymphocyte such as a T cell. The present methods advantageously allow high delivery yield of a cargo, such as a m RNA, into imm une cells, e.g. T cells.

Methods of obtaining imm une cells, such as T cells, from an animal or human subject are well known in the art.

I n embodiments, the cell may be an isolated cell or cultured cell.

The term “isolated” as used throughout this specification with reference to a particular component generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment. More particularly, the term “isolated” as used herein in relation to cells or tissues denotes that such cells or tissues do not or no longer form part of a plant, an animal or human body.

Isolated cells or tissues may be suitably cultured or cultivated in vitro. The terms “culturing” or “cell culture” are com mon in the art and broadly refer to maintenance of cells and potentially expansion (proliferation, propagation) of cells in vitro. Typically, plant cells or animal cells, such as mammalian cells, such as human cells, are cultured by exposing them to (i.e., contacting them with) a suitable cell culture medium in a vessel or container adequate for the purpose (e.g., a 96-, 24- , or 6- well plate, a T-25, T-75, T-150 or T-225 flask, or a cell factory) , at art-known conditions conducive to in vitro cell culture.

I n embodiments, the concentration of the cells in the mixture may be about 10 ² to about 10 ¹⁰ cells per m illilitre (m l) and more preferably about 10 ³ to about 10 ⁹ cells/ml, such as for example about 10 ⁴ to about 10 ⁸ cells/ml or about 10 ⁵ to about 10 ⁸ cells/ml. The cells are preferably present in the mixture in an aqueous solution such as in a cell culture medium or a suitable transfection buffer solution.

I n embodiments, delivering (or the delivery of) a cargo into a cell may be transfecting (or the transfection of) a cargo into a cell. I n embodiments, the methods as taught herein may thus be for transfecting or the transfection of a cargo into a cell.

The term “transfection” refers to the process of introducing a nucleic acid into an animal cell.

I n embodiments, delivering (or the delivery of) a cargo into a cell may be transforming (or the transformation of) a cargo into a cell. I n embodiments, the methods as taught herein may thus be for transform ing or the transformation of a cargo into a cell.

The term “transformation” refers to the process of introducing a nucleic acid into an non-animal eucaryotic cell such as a plant cell.

I n embodiments of the methods as taught herein, the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, e.g. a liposome or a solid lipid particle, and combinations thereof.

The term “based” as used in the context of the material of the organic particle as defined above is to be understood as a particle that predom inantly comprises or is made of said material. I n other words, said protein -based particle is to be understood as a particle that mainly comprises or completely consists of one or more proteins or peptides. A lipid-based or “lipid particle” may be used interchangeably herein and refer to particles comprising, consisting essentially of, or consisting of one or more lipids.

The terms “polymer-based particle” or “polymer particle” may be used interchangeably herein and refer to particles comprising, consisting essentially of, or consisting of one or more polymers.

As used herein, the term “polymer” refers to a macromolecule composed of repeating subunits or monomers. Preferably, each monomer comprises carbon and one or more additional elements such as hydrogen, oxygen or nitrogen. The term encompasses both synthetic and natural polymers but excludes biopolymers such as nucleic acids and proteins. I n embodiments, the organic particle may be a polymer-based particle.

I n embodiments, the polymer-based particle is not a carbon-based particle such as a carbon-based m icroparticle or carbon-based nanoparticle. I n embodiments, the polymer-based particle is not an allotrope of carbon. I n embodiments, the polymer- based particle is not a particle selected from the group consisting of a graphene particle (e.g. nanoribbon) , graphene oxide particle, carbon nanotube, carbon dot, fullerene, graphite, or diamond, or a combination thereof such as a carbon nanobud. I n embodiments, the polymer is not consisting of carbon. I n embodiments, the polymer is not an allotrope of carbon such as graphene. I n embodiments, the polymer is not a solid form of carbon such as diamond. I n embodiments, the polymer is not a crystalline form of carbon such as graphite.

I n embodiments of the methods or products as taught herein, the polymer-based particle may comprise or consist of poly( DL-lactic-co-glycolic acid) (PLGA) , poly(lactic acid) (PLA) , polycaprolactone (PCL) , ethyl cellulose, cellulose acetophthalate, cellulose, polyvinyl alcohol, polyethylene glycol, gelatine, collagen, silk, alginate, hyaluronic acid, dextran, starch, polycarbonate, polyacrylate, polystyrene, m ethoxy-PEG-polylactide, poly(alkyl cyanoacrylate) (PACA) , poly(D,L- lactide-co-glycolide (PLGH) , poly(allylamine hydrochloride) , or a polyoxazoline. For example, PLGA-based I CG nanoparticles (PLGA-ICG NPs) may be prepared as described in Saxena et al., 2004, I nt J Pharm , 278(2) :293-301 .

Suitable examples of PACA include poly(butyl cyanoacrylate) (PBCA) , poly(octyl cyanoacrylate) (POCA) , and poly (ethyl 2-cyanoacrylate) (PECA) .

I n embodiments, the polymer-based particle may comprise or consist of one or more clinically approved polymers, such as methoxy-PEG-polylactide, poly(alkyl cyanoacrylate) (PACA) , poly(D,L-lactide-co-glycolide (PLGH) , and poly(allylamine hydrochloride) .

I n embodiments, the organic particle may be a protein-based particle. Protein-based particles are highly stable, biocompatible, and biodegradable, thereby allowing the delivery of a cargo into a cell without any toxicity to the cell.

The terms “protein-based particle” or “protein particle” may be used interchangeably herein and refer to particles comprising, consisting essentially of, or consisting of one or more proteins. Protein-based particles include protein-based nanoparticles (PNPs) such as non-viral particles based on ferritin, heat shock proteins (Hsp) , DNA-binding proteins from starved cells (Dps) , encapsulin, the E2 protein of pyruvate dehydrogenase, lumazine synthase, vault proteins; and virus-like particles (VLPs) .

I n embodiments, the protein may be an albumin such as human serum albumin (HSA) . For example, human serum albumin ICG nanoparticles (HSA-I CG NPs) may be prepared as described in Sheng et al. , 2014, ACS Nano, 8(12) : 12310-22.

I n embodiments, the organic particle may be a protein-based particle comprising one or more clinically approved proteins such as albumin-bound nanoparticles (e.g. Abraxane) . I n embodiments, the organic particle may be a protein-based particle comprising or one or more clinically approved protein-based drugs, such as engineered protein combining I L-2 and diphtheria toxin (e.g. Ontak) ; PEG- asparaginase (e.g. Oncaspar) ; PEG-filgrastim (PEGylated granulocyte colonystimulating factor (GCSF) protein) (e.g. Neulasta) ; PEGylated porcine-like uricase (e.g. Krystexxa) ; PEGylated factor VI I I (e.g. ADYNOVATE) ; PEGylated I FN alpha-2a protein (e.g. Pegi ntron) ; PEGylated adenosine deaminase enzyme (e.g. Adagen/pegademase bovine) ; PEGylated antibody fragment (e.g. Cimzia/certolizumab pegol) ; random copolymer of L-glutamate, L-alanine, L-lysine and L-tyrosine (e.g. Copaxone/Glatopa) ; PEGylated anti-vascular endothelial growth factor (VEGF) aptamer (e.g. Macugen) , methoxy polyethylene glycol-epoetin beta (e.g. Mircera) ; PEGylated GCSF protein (e.g. Neulasta) ; PEGylated I FN alpha-2a protein (e.g. Pegasys) .

The terms “lipid-based particle” or “lipid particle” may be used interchangeably herein and refer to particles comprising, consisting essentially of, or consisting of one or more lipids. Lipid-based articles are biocompatible, and biodegradable, thereby allowing the delivery of a cargo into a cell without any toxicity to the cell. The lipid-based particles may be liposomes or may be solid lipid particles.

I n embodiments, the lipid-based particles may comprise a natural, a bacterial or a synthetic lipid. I n embodiments, the lipid-based particles may comprise an anionic lipid, neutral lipid, cationic lipid, ionizable lipid or a sterol.

Suitable examples of anionic lipids include phosphatidylserine (PS) and phosphatidylglycerol (PG) .

Suitable examples of neutral lipids include prostaglandins, eicosanoids, glycerides, glycosylated diacyl glycerols, oxygenated fatty acids, very long chain fatty acids (VLCFA), palmitic acid esters of hydroxystearic acid (PAHSA), N-acylglycine (NAGIy), and prenols.

Suitable examples of cationic lipids include multivalent cationic lipids; 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA); ethylphosphocholines (EPC); dim ethyldioctadecy lam monium (DDAB) ; N1 -[2-((1 S)-1 -[ (3- am i nopropyl) ami no] -4- [ di (3- am in o- propyl) am in o] butylcarboxam ido) ethyl] - 3 , 4- d i [ oleyl oxy] - benzamide (MVL5); pH sensitive lipids; 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP); 30- [N-(N',N'-dim ethylaminoethane) -carbamoyl] cholesterol (DC- Cholesterol) ; N4- Cholesteryl-spermine (GL67); 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA); Dlin-MC3-DMA (MC3); DLinDAP; DLinDMA; DLinKDMA; or DLinKC2-DMA.

Such lipids are commercially available from Avanti Polar Lipids (Alabama, USA). For instance, a suitable multivalent cationic lipid is (N1-[2-((1S)-1-[(3- am i nopropyl) ami no] - 4- [di(3- ami no- propyl) ami no] butylcarboxam ido) ethyl] -3,4- di[oleyloxy]-benzamide). Examples of ethyl PC include 1 ,2-dilauroyl-sn-glycero-3- ethylphosphocholine (chloride salt) (12:0 EPC Cl salt); 1 ,2-dimyristoyl-sn-glycero- 3-ethylphosphocholine (chloride salt) (14:0 EPC Cl salt); 1 ,2-dipalmitoyl-sn-glycero- 3-ethylphosphocholine (chloride salt) (16:0 EPC Cl salt); 1 ,2-distearoyl-sn-glycero- 3-ethylphosphocholine (chloride salt) (18:0 EPC Cl salt); 1 ,2-dioleoyl-sn-glycero-3- ethylphosphocholine (chloride salt) (18:1 EPC Cl salt); 1 -palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine (chloride salt) (16:0-18:1 EPC Cl salt); and 1,2- dimyristoleoyl-sn-glycero-3- ethylphosphocholine (Tf salt) (14:1 EPC Tf salt) .

Examples of pH sensitive lipids include N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis( oleoyl oxy ) propan- 1 - aminium (DOBAQ) ; 1 ,2-distearoyl-3-dim ethylammonium - propane (18:0 DAP); 1 ,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP); 1 ,2-dimyristoyl-3-dimethylam monium-propane (14:0 DAP); 1 ,2-dioleoy I-3- dimethylammonium-propane (18:1 DAP or DODAP))

Suitable examples of ionizable lipids include Dlin-MC3-DMA (MC3); DLinDAP; DLinDMA; DLinKDMA; or DLinKC2-DMA.

Suitable examples of sterols include oxysterols, natural sterols, bile acids, cholesteryl esters, glycosylated sterols, or oxidized sterols.

Methods for the preparation of lipid-based particles are known in the art. In embodiments, the lipid-based particles may be MC3-based lipid particles (Patel et al., 2019, J. Control. Release, 303, 91-100). Liposomes encapsulating ICG (Lip- ICG) may be prepared as described in Lajunen et al., 2018, J. Control. Release 284, 213-

223.

I n embodiments of the methods or product as taught herein, the liposomes may be prepared by methods known in the art such as thin film rehydration method (e.g. including extrusion or sonication for the incorporation of the light absorbing molecule into the core of the liposomes) ; microfluidic m ixing; or injection method, e.g. including dropping a solvent containing lipids and light absorbing molecules in a physiological buffer.

For example, liposomes may be prepared by m ixing 1 ,2- Dioleoy I-3- trimethylam monium propane (DOTAP) , 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolam ine (DOPE) , and 1 ,2-distearoyl-sn-glycero-3- ph osph oet h anolam ine-N-[ am in o( polyethylene glycol) -2000] (DSPE-PEG(2000)

Amine) at a certain molar ratio. A light absorbing molecule such as I CG may then be incorporated by an extrusion and/or sonication step (tip/bath sonication) . Purification may be obtained by ultracentrifugation and/or dialysis.

I n embodiments of the methods or product as taught herein, the solid lipid particles may be prepared by methods known in the art such as high shear homogenization (e.g. hot or cold homogenization) ; ultrasonication/high speed homogenization ; solvent em ulsification/evaporation ; m icro emulsion based solid lipid particles preparation ; solid lipid particles preparation using supercritical fluid; spray drying method; and double em ulsion method.

I n embodiments, the photoresponsive organic particle may be a polymer-based particle, a protein-based particle, a lipid-based particle, or a combination of two or more of a polymer-based particle, a protein-based particle, a lipid-based particle. The polymer-based particle, protein-based particle, lipid-based particle, or combination thereof may itself have photoresponsive properties such as a polydopam ine particle. Alternatively, the polymer-based particle, protein-based particle, lipid-based particle, or combination thereof may comprise a light absorbing molecule (having photoresponsive properties) , thereby providing the polymer-based particle, protein-based particle, lipid-based particle, or combination thereof with photoresponsive properties. Both types of photoresponsive organic particles can be prepared using precursor molecules that are clinically approved. The term “photoresponsive”, “photosensitive”, “light sensitising” may be used interchangeably and refer to the capacity to respond to electromagnetic radiation, such as e.g. visible light.

I n embodiments of the methods as taught herein or products as taught herein (including the photoresponsive organic particles as taught herein) , the photoresponsive organic particle may be a photoresponsive polymer-based particle, a photoresponsive protein-based particle, a photoresponsive lipid-based particle, or a combination thereof. Such photoresponsive organic particles may be prepared using clinically approved precursors, thereby facilitating clinical transition of the delivery methods as taught herein for the production of engineered therapeutic cell products, such as CAR-T cells.

I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be a photoresponsive polymer-based particle. I n embodiments, the photoresponsive organic particle may be a photoresponsive polymer-based particle selected from a polydopamine (PD) particle, a poly(N-phenylglycine) (PNPG) particle, a poly-2-phenyl-benzobisthiazole (PPBBT) particle, a porphyrin particle, a phthalocyanine particle, or a polypyrrole particle. I n embodiments, the photoresponsive organic particle may comprise or consist of polydopam ine, poly(N- phenylglycine) , poly-2-phenyl-benzobisthiazole, porphyrin, phthalocyanine or polypyrrole. I n embodiments, the photoresponsive organic particle may be prepared (produced or synthetised) from a clinically approved monomer, such as dopam ine hydrochloride, thereby facilitating clinical transition of the methods as taught herein for the production of engineered therapeutic cell products, e.g. CAR-T cells.

I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be a polymer-based particle, a protein-based particle, a lipid- based particle (e.g. liposome or solid lipid particle) , or a combination thereof comprising a light absorbing molecule. I n embodiments, the photoresponsive organic particle may be a polymer-based particle comprising a light absorbing molecule. I n embodiments, the photoresponsive organic particle may be a protein-based particle comprising a light absorbing molecule. I n embodiments, the photoresponsive organic particle may be a lipid-based particle comprising a light absorbing molecule. I n embodiments, the photoresponsive organic particle may be a solid lipid particle comprising a light absorbing molecule. I n embodiments, the photoresponsive organic particle may be a combination of two or more of a polymer-based particle, a proteinbased particle, a lipid-based particle comprising a light absorbing molecule. Such photoresponsive organic particles may be prepared using clinically approved molecules, thereby facilitating clinical transition of the delivery methods as taught herein for the production of engineered therapeutic cell products, such as CAR-T cells.

In embodiments of the methods or products as taught herein, the photoresponsive organic particle may be a polymer-based particle, a protein-based particle, a lipid particle loaded with or functionalized with a light absorbing molecule.

In embodiments, the light absorbing molecule may be grafted on a particle, such as a nanoparticle or a microparticle. For instance, the light absorbing molecule may be grafted on a particle by ‘click-chemistry’ at the surface of the particle (e.g. at the end of polymer chains such as at the distal end of poly(ethylene) glycol chains or hyaluronic acid chains). For instance, the grafting of a light absorbing molecule may occur at the end of PEG chains that are grafted on the particles.

In embodiments, the light absorbing molecule may be encapsulated in a particle, such as a nanoparticle or a microparticle. In embodiments, the light absorbing molecule may be encapsulated in a particle by physical or chemical encapsulation. For instance, physical encapsulation of ICG in liposomes may be performed by adding ICG during the rehydration of the lipids. For human serum albumin-ICG particles, the chemical encapsulation may be performed by reacting ICG with the disulphide bonds of HSA.

Examples of photoresponsive protein-based particles include PLGA-based ICG particles.

Examples of photoresponsive protein-based particles include heptamethine dye (CySCOOH) coupled to human serum albumin (HSA) particles; HSA coupled to Ce6 (HSA-Ce6); HSA coupled to squaraine (SQ) dye (HAS-SQ); HSA-IR825 complexes; loading of zinc hexadecafluorophthalocyanine (ZnF16Pc) in Cys-Asp-Cys-Arg-Gly Asp- Cys-Phe-Cys (RGD4C)-modified ferritins (RFRTs); methylene blue encapsulation in apoferritin particles; IR820 dye-loaded ferritin particles; particles consisting of cowpea chlorotic mottle virus (CCMV) protein with a water soluble zinc Pc (ZnPc); albumin coupled to 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrine (mTHPP) and/or 5,10,15,20-tertrakis(m-hydroxyphenyl)chlorin (mTHPC); hematoporphyrin- linked albumin nanoparticles (HP-ANP); polypyrrole (PPy) complexed albumin nanoparticles; polypyrrole (PPy) complexed with albumin and Ce6 particles; Poly-L- Lysine (PLL) complexed with albumin particles containing Ce6; Poly-L-Lysine (PLL) complexed with albumin particles containing protoporphyrin IX; Poly-L-Lysine (PLL) complexed with albumin particles containing verteporfin; hydrogels containing meso- tetra-(N-methyl-4-pyridyl) porphine tetrachloride (TMPyP); and HSA-IR780 particles. Examples of photoresponsive liposomes include for instance liposomes comprising ICG.

Examples of photoresponsive solid lipid particles include for instance solid lipid particles containing hydrophobic I R-780 Dye; solid lipid particles containing ICG; and solid lipid particles containing doxorubicin.

In embodiments of the methods or products as taught herein, the light absorbing molecule may be a molecule selected from the group consisting of a light absorbing dye, a naturally occurring light absorber, and a synthetic light absorber.

The light absorbing molecule may be lipophilic, hydrophilic, or amphiphilic. The light absorbing molecule may be a lipophilic compound embedded into the lipid membrane or lipid core of an organic particle. The light absorbing molecule may be a hydrophilic compound encapsulated into the aqueous core of an organic particle. The light absorbing molecule may be an amphiphilic compound embedded into the lipid membrane or lipid core of an organic particle and/or encapsulated into the aqueous core of the organic particle.

The light absorbing dye may be may be a natural dye or a synthetic dye.

The light absorbing dye may be selected from the group consisting of an ANEPdye; a 7-nitrobenz-2-oxa-1 ,3-diazole-4-yl-labeled phospholipid; 1 - ol eoy I - 2- [ 6 - [ ( 7- n it ro- 2-1 ,3-benzoxadiazol-4-yl)am ino] hexanoyl] - 3- trimet hy lam mon ium propane

(fluorescent DOTAP); bis-( 1 ,3-dibutylbarbitu ric acid)trimethine oxonol (DiBAC4(3)); N-(3-Triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide; ethidium bromide; propidium iodide; chlorin e6 (Ce6); purpurin-18 (P18); and heptamethine dye (CySCOOH).

Examples of ANEP dyes include Di-4-ANEPPS; Di-4-ANEPPDHQ; Di-3- ANEPPDHQ; and Di-8-ANEPPS such dyes are commercially available from ThermoFisher Scientific. Examples of 7-nitrobenz-2-oxa-1 ,3-diazole-4-yl-labeled phospholipids include [2-(4- nitro-2,1 ,3-benzoxadiazol-7-yl) aminoethyl] trim ethylammonium (NBD-TMA) ; N-(7- Nitrobenz-2-Oxa-1 , 3 - Di azol- 4- y I ) - 1 ,2- Dih exadecan oyl-sn-Glycero-3- Phosphoethanolamine, Triethylammonium Salt) (NBD-PE) ; and NBD-DOPE.

The light absorbing dye may be selected from the group consisting of an azo dye, an arylmethane dye, a cyanine dye, a thiazine dye, a xanthene dye, a carbocyanine dye, and an am inostyryl dye.

Examples of azo dyes include Trypan Blue (Membrane Blue, Vision Blue, CAS Number: 72-57-1 ) and Janus green B (Diazine Green S, Union Green B, CAS Number: 2869-83-2) .

Examples of arylmethane dyes include Gentian violet (Crystal violet, Methyl violet 10B, Hexamethyl pararosaniline chloride, CAS Number: 548-62-9) ; Bromophenol Blue (CAS Number: 1 15-39-9) ; Patent blue (Blueron, CAS Number: 3536-49-0) ; Brilliant Blue (Acid Blue, Coomassie Brilliant Blue, Brilliant Peel, CAS Number: 6104- 59-2) ; Light Green (Light Green SF, Light Green SF Yellowish, CAS Number: 5141 - 20-8) ; and Fast Green (Fast Green FCF, Food green 3, FD&C Green No. 3, Green 1724, Solid Green FCF, CAS Number: 2353-45-9) .

Examples of cyanine dyes include I ndocyanine Green (Cardiogreen, Foxgreen, Cardio-Green, Fox Green, I C Green, CAS Number: 3599-32-4) and I nfracyanine Green. I nfracyanine Green (IfCG) is a green dye with the same chem ical formula and similar pharmacologic properties as I CG. IfCG dye possesses two pharmacologic differences when compared to I CG. First, If I CG contains no sodium iodine, which m ust be added to I CG during the dye synthesis. Second, the presence of the sodium iodine in the I CG solution necessitates dilution in water, resulting in a hypotonic solution.

Examples of thiazine dyes include Methylene blue (Methylthioninium chloride, CAS Number: 61 -73-4) and Toluidine blue (CAS Number: 92-31 -9) .

Examples of xanthene dyes include Fluorescein Sodium (CAS Number: 518-47-8) ; Rose Bengal (CAS Number: 4159-77-7) ; and Rhodamine 6G (Rhodam ine 590, Rh6G, C.l . Pigment Red 81 , C.l . Pigment Red 169, Basic Rhodamine Yellow, C.l . 45160, CAS Number: 989-38-8) .

Examples of carbocyanine dyes include Dil (Cat. no. D282, D391 1 , N22880, I nvitrogen) ; DiO (Cat. no. D275, I nvitrogen) ; DiD (Cat. no. D307, D7757, I nvitrogen) ; DiR (Cat. no. D12731 , I nvitrogen) , and derivatives thereof. Derivatives of carbocyanine dyes include Dil C12(3) (Cat. no. D383, I nvitrogen) ; Di I C16(3) (Cat. no. D384, I nvitrogen) ; DiOC16(3) (Cat. no. D1 125, I nvitrogen) ; unsaturated derivatives, e.g. m ono-unsaturated or di-unsaturated derivatives, of Dil and DiO, such as A9 - Dil ; (Cat. No. D3886, I nvitrogen) ; sulfonated derivatives of Dil and DiO; and CM-Dil (a thiol-reactive Dil derivative) .

Examples of aminostyryl dyes include DiA (Cat. no. D3883, I nvitrogen) , and 4-Di- 10-ASP (Cat. no. D291 , I nvitrogen) , and di-unsaturated derivatives of DiA (Cat. no. D7758, I nvitrogen) .

I n embodiments of the methods or products as taught herein, the light absorbing dye may be selected from the group consisting of indocyanine green (I CG) , trypan blue, Janus green B, gentian violet, bromophenol blue, patent blue, brilliant blue, light green, fast green, infracyanine green, methylene blue, toluidine blue, fluorescein sodium , rose Bengal, rhodamine 6G, DiD, DiO, Dil , DiA, DiR, and derivatives of Dil , DiO, or DiA. I n embodiments of the methods or products as taught herein, the light absorbing dye may be selected from the group consisting of indocyanine green (I CG) , trypan blue, Janus green B, gentian violet, bromophenol blue, patent blue, brilliant blue, light green, fast green, infracyanine green, methylene blue, toluidine blue, fluorescein sodium , rose Bengal, and rhodamine 6G. Such light absorbing dyes are advantageously approved for clinical use.

Other light absorbing molecules which have been clinically approved include porfimer sodium (e.g. sold under commercial name Photofrin); 6-Aminolevulinic acid (e.g. sold as Levulan) ; verteporfine (e.g. sold as Visudyne) ; temoporfin (e.g. sold as Foscan) ; sulfonated aluminum phthalocyanine (e.g. sold as Photosense) ; methyl am inolevulinate (MAL) (e.g. sold as Metvix) ; talaporfin or mono-L-aspartyl chlorin e6 (e.g. sold as Laserphyrin) .

Other light absorbing molecules include for example pheophorbide; phthalocyanine derivatives (e.g. zinc (I I ) phthalocyanine, silicon phthalocyanine 4) ; cyanine I R-768; hypericin; hypocrellin A; C60 or C70 fullerene cage; and pheophorbide A (PheA) .

The naturally occurring light absorber may be selected from the group consisting of hemoglobin, cytochrome C, porphyrin, and pigments. The pigment may be for example melanin, rhodopsin, photopsins, iodopsin, chlorophyll a, chlorophyll b, phycoerythrin, -carotene, phycocyanin, or allophycocyanin.

The synthetic light absorber can be selected from the group consisting of polydopamine, poly(N-phenylglycine, poly-2-phenyl-benzobisthiazole, porphyrin, phthalocyanine, or polypyrrole. The term “delivery yield” as used herein refers to the ratio of the quantity of living (viable) cells comprising the cargo after perform ing the method as taught herein (e.g. the quantity of living cells comprising the cargo as detected after the delivery method) relative to the quantity of living (viable) cells before perform ing the method as taught herein (e.g. the quantity of living cells as detected before the delivery method) .

The delivery yield expressed as a percentage (%) can be determined by multiplying the viability of the cells after perform ing the method as taught herein (e.g. expressed as a percentage) with the efficiency of the method as taught herein (e.g. expressed as a percentage) , followed by dividing the resulting value by 100.

Delivery yield (%) = { [cell viability (%)] x [efficiency (%)]} /100

The viability of cells after performing the method as taught herein (%) may be determined by dividing the quantity, such as number, of viable cells obtained after perform ing the method as taught herein by the quantity, such as number, of (total) viable cells before performing the method as taught herein, followed by multiplying the resulting value by 100.

The efficiency of the method as taught herein (%) may be determ ined by dividing the quantity, such as number, of viable cells comprising the cargo obtained after performing the method as taught herein by the quantity, such as number, of (total) viable cells obtained after perform ing the method as taught herein, followed by m ultiplying the resulting value by 100.

The delivery yield (%) = { [ (number of viable cells after delivery method/number of viable cells before delivery method) x100] x [ (number of viable cells comprising cargo after delivery method/number of viable cells after delivery method) x 100]} / 100

I n embodiments of the methods as taught herein, the delivery yield (e.g. maximal delivery yield) when using photoresponsive organic particles having the largest dimensions of about 100 nm to about 550 nm , more preferably between 150 and 550 nm , more preferably between 100 and 500 nm , more preferably between 200 and 400 nm , more preferably 200 and 300 nm , more preferably of between 225 and 275 nm , more preferably between 230 and 260 nm , more preferably between 240 and 255 nm, such as 250 nm or wherein said particle size is between 150 and 550 nm, more preferably between 200 and 550 nm, more preferably between 300 and 550 nm, more preferably 400 and 550 nm, more preferably of between 450 and 550 nm, such as 500 nm may be enhanced (i.e., increased) by at least about 5% relative to (i.e., compared with) the delivery yield (e.g. maximal delivery yield) when using photoresponsive organic particles having the largest dimensions outside this range (e.g. of about 1000 nm or larger). In embodiments of the methods as taught herein, the delivery yield (e.g. maximal delivery yield) when using photoresponsive organic particles having the largest dimensions of about 100 nm to about 550 nm, more preferably between 150 and 550 nm, more preferably between 100 and 500 nm, more preferably between 200 and 400 nm, more preferably 200 and 300 nm, more preferably of between 225 and 275 nm, more preferably between 230 and 260 nm, more preferably between 240 and 255 nm, such as 250 nm or wherein said particle size is between 150 and 550 nm, more preferably between 200 and 550 nm, more preferably between 300 and 550 nm, more preferably 400 and 550 nm, more preferably of between 450 and 550 nm, such as 500 nm may be at least enhanced (i.e., increased) by at least about 10%, by at least about 15%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 35%, by at least about 40%, by at least about 45%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by at least about 100%, by at least about 200%, by at least about 300%, by at least about 400%, or by at least about 500%, relative to (i.e., compared with) the delivery yield (e.g. maximal delivery yield) when using photoresponsive organic particles having the largest dimensions outside this range.

For example, an increase may encompass an increase of a first value of the delivery yield by, without limitation, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%, relative to a second value of the delivery yield with which a comparison is being made.

For instance, in a first example, an increase may encompass an increase of a first value of the delivery yield (e.g., 10% delivery yield) by 20% (i.e.2% / 10% x 100%) relative to a second value of the delivery yield (e.g., 12% delivery yield) with which a comparison is being made. In a second example, a deviation may encompass an increase of a first value of the delivery yield (e.g., 10% delivery yield) by 200% (i.e. 20%/10% x 100%) relative to a second value of the delivery yield (e.g., 30% delivery yield) with which a comparison is being made.

Accordingly, also provided herein is a photoresponsive organic particle, wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, preferably a polydopamine particle and wherein the particle size is of between 100 nm to 550 nm , more preferably between 150 and 550 nm , more preferably between 100 and 500 nm , more preferably between 200 and 400 nm , more preferably 200 and 300 nm , more preferably of between 225 and 275 nm , more preferably between 230 and 260 nm , more preferably between 240 and 255 nm , such as 250 nm or wherein said particle size is between 150 and 550 nm , more preferably between 200 and 550 nm , more preferably between 300 and 550 nm , more preferably 400 and 550 nm , more preferably of between 450 and 550 nm , such as 500 nm . As extensively demonstrated in the example section, such photoresponsive organic particles advantageously allow efficient delivery of a cargo such as nucleic acids including m RNA into a cell, while keeping satisfactory cell viability, thereby resulting in high delivery yield of a cargo into the cell.

The photoresponsive organic particles may comprise individual particles or a group, agglomerate, or cluster of two or more particles positioned close to or in contact with each other.

I n embodiments, the photoresponsive organic particles may be present as individual particles, for instance in an aqueous solution, such as in a cell culture medium .

I n embodiments, the photoresponsive organic particles may comprise a group, agglomerate, or cluster of two or more particles, for instance in an aqueous solution, such as in a cell culture medium .

The term “particle” or “nanoparticle” as used herein refers to a particle or a group, agglomerate, or cluster of two or more particles having dimensions (more particularly the largest dimensions of the particles) of about 100 nm to about 550 nm . The dimensions of a particle, for example a width, height or diam eter of a particle, can be determined using Transm ission Electron Microscopy (TEM) , Scanning Electron Microscopy (SEM) , Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) or atomic force microscopy (AFM) . I n a preferred embodiment, said particle size is determ ined by using Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) .

I n embodiments, the methods as taught herein may comprise contacting the cell with one type of photoresponsive organic particles or a combination of different photoresponsive organic particles, for example photoresponsive organic particles having a different size, a different composition and/or a different shape.

The particles may have any shape. For example, the particles may be spherical, elliptical, rod-like shaped, pyramidal, branched, or may have an irregular shape.

I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be an elliptical particle, rod-like shaped particle, pyramidal particle, branched particle, or may be an irregular shaped particle. The size of such particles is preferably defined by the equivalent spherical diameter (also referred to as the equivalent volume diameter) . The equivalent spherical diameter (or ESD) of an irregularly shaped object is the diameter of a sphere of equivalent volume.

The particles may have a spherical shape. I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be a spherical particle. I n embodiments, the photoresponsive organic particle may be a spherical particle having an average diameter of about 100 nm to about 550 nm . Such photoresponsive organic particle advantageously allow efficient delivery of a cargo such as large macromolecules into a cell.

I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be functionalized on the surface such as coated on the surface. I n embodiments, the photoresponsive organic particle may be functionalized, such as coated, with one or more compounds selected from the group consisting of album in, polyethyleneimine (PEI ) , polyvinylpyrrolidone (PVP) , polyethylene glycol (PEG) , poly(diallyldimethylammonium chloride) (PDDAC) , poly(allylamine hydrochloride) (PAH) , polyam idoam ine (PAA) , poly(amino-co-ester) (PAE) , poly[2- (N,N-dimethylam ino)ethyl methacrylate] (PDMAEMA) , hyaluronic acid (HA) , gelatin, polyglycerol, a cyclodextrin (CD) , dextran, cellulose, silica, polyoxazoline, su If obet ai n e- si Ian e (SBS) , a cationic lipid, a neutral lipid, an anionic lipid, chitosan, and poly-L-Lysine. Advantageously, photoresponsive organic particles which are further functionalized, such as polydopamine particles functionalized with album in, allow to improve colloidal stability without interfering with or even improving the particles ability to adhere to a barrier of the cell, such as the cell membrane or cell wall.

I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be a polydopamine particle. I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be a polydopamine particle coated with album in. Such photoresponsive organic particles may advantageously be biocompatible and biodegradable, and have sufficient colloidal stability, for example in a suitable cell medium or transfection buffer solution.

I n embodiments, the photoresponsive organic particles are capable of binding to the cells. I n embodiments, the photoresponsive organic particles may be capable of binding to (or accum ulating at) the cells after contacting the cell with the photoresponsive organic particles. The binding of the photoresponsive organic particles to the cell may be binding by a covalent binding or a non-covalent interaction such as an electrostatic or hydrophobic interaction.

I n embodiments, the methods as taught herein may comprise contacting the cell with the photoresponsive organic particle, thereby inducing binding of the photoresponsive organic particle to (accum ulation of the photoresponsive organic particle at) the cell, in particular to a barrier of the cell, such as to the cell wall or cell membrane of the cell.

I n embodiments of the methods and products as taught herein, the photoresponsive organic particle may be capable of forming vapour nanobubbles the cell, in particular at a barrier of the cell, when irradiated. When using the photoresponsive organic particle in accordance with embodiments of the invention, the photoresponsive organic particle may form vapour nanobubbles at the cell, in particular at a barrier of the cell, when irradiated.

I n embodiments, the methods as taught herein may comprise irradiating the mixture of the cell, the cargo, and the photoresponsive organic particle with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and allowing influx of the cargo into the cell. I n embodiments, the methods may comprise irradiating the photoresponsive organic particle bound to at least part of the cell, thereby form ing vapour nanobubbles at a barrier of the cell and inducing permeabilization of the cellular barrier.

I n embodiments of the methods or products as taught herein, the photoresponsive organic particle may be biodegradable. I n embodiments, the photoresponsive organic particle may be biocompatible. I n embodiments, the photoresponsive organic particle may be biodegradable and biocompatible. I n embodiments, the photoresponsive organic particle may comprise or consist of clinically approved components, such as polymers, proteins, lipids, solid lipids, and/or light absorbing molecules. Advantageously, this renders the photoresponsive organic particles biocompatible and/or biodegradable.

I n embodiments of the methods as taught herein, the photoresponsive organic particles are not coupled to a solid support, e.g. the photoresponsive organic particles are not coupled (e.g. grafted) to or enclosed (e.g. embedded) in a solid support or solid material. I n embodiments of the methods as taught herein, the photoresponsive organic particles are not enclosed (e.g. embedded) in a non-porous structure such as a polymer sheet or polymer foil. I n embodiments of the methods as taught herein, the photoresponsive organic particles are not enclosed (e.g. embedded) in a porous polymer structure such as structures comprising fibres (for example polymer fibres) , structures comprising particulates (for example polymer particulates) , structures comprising a combination of fibres and particulates (for example a combination of polymer fibres and/or polymer particulates) and structures comprising foam (for example polymer foam) . I n embodiments of the methods as taught herein, the photoresponsive organic particles are in solution.

I n embodiments, the m ixture may comprise the photoresponsive organic particles in a concentration of about 10 ⁵ to about 10 ¹¹ particles per m illilitre (ml) and more preferably about 106 to about 1010 particles/ml, such as for example about 107 to about 109 particles/ml, e.g. to balance efficient permeabilization with cell viability. The mixture preferably comprises the photoresponsive organic particles in an aqueous solution, such as in a cell culture medium or a suitable transfection buffer solution, in which the photoresponsive organic particles have sufficient colloidal stability.

The photoresponsive organic particles may be comprised in a composition or form ulation such as a pharmaceutical formulation or kit of parts, as will be described further herein. The composition may comprise the photoresponsive organic particles in a concentration ranging from about 108 to about 10 ¹⁵ particles/m I, such as for example about 10 ¹⁰ to about 10 ¹² particles/m I.

I n embodiments of the methods as taught herein, the cell, the photoresponsive organic particle, and the cargo may be contacted in the mixture in a concentration of about 10 ² to about 10 ¹⁰ cells/ml, about 10 ⁵ to about 10 ¹¹ particles/m I, and about 0.001 to about 10 mg/ml (i.e. pg/pl) cargo/ml (with ml referring to the volume of the mixture) .

I n embodiments of the methods as taught herein, the cargo may be a nucleic acid such as a plasmid DNA encoding a chimeric antigen receptor (CAR) or a m RNA encoding a CAR, and the cells may be (naive) T cells. I n embodiments of the methods as taught herein, the cargo may be a nucleic acid such as a plasmid DNA encoding a CAR or a m RNA encoding a CAR; the photoresponsive organic particle may be a polydopam ine particle, preferably a polydopam ine particle coated with albumin; and the cells may be T cells. The present methods advantageously allow the delivery of desired genetic constructs, e.g. encoding a chimeric antigen receptor, into human T cells to generate chimeric antigen receptor T cells.

The term “chimeric antigen receptor” or “CAR” (also known as chimeric imm unoreceptors, chimeric T cell receptors or artificial T cell receptors) refers to a receptor protein that has been engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigenbinding and T-cell activating functions into a single receptor.

I n embodiments of the methods as taught herein, the cargo may be a m RNA encoding a CAR and the cells may be T cells. I n embodiments of the methods as taught herein, the cargo may be a m RNA encoding a CAR; the photoresponsive organic particle may be a polydopam ine particle, preferably a polydopam ine particle coated with album in ; and the cells may be T cells. The present methods advantageously allow delivery and transient expression of m RNA into T cells, thereby providing control over the duration of expression without any risks for unintended m utations by genom ic integration.

I n embodiments, the methods comprise contacting the cell with the one or more photoresponsive organic particles. I n embodiments, the methods comprise contacting the cell with the one or more photoresponsive organic particles and the cargo.

The term “contact” or “contacting” as used herein means bringing one or more first components (such as one or more molecules, biological entities, cells, or materials) together with one or more second components (such as one or more molecules, biological entities, cells, or materials) in such a manner that the first component(s) can - if capable thereof - interact with such as bind or modulate the second component(s) or that the second component(s) can - if capable thereof - interact with such as bind or modulate the first component(s) . Such modulation may occur either directly, i.e., by way of direct interaction between the first and second component(s) ; or indirectly, e.g., when the first component(s) interact with or modulate one or more further component(s) , one or more of which in turn interact with or modulate the second component(s) , or vice versa. The term “contacting” may depending on the context be synonymous with “exposing”, “bringing together”, “m ixing”, “reacting”, “treating”, or the like.

I n embodiments, the contacting step (e.g. in suspension) may comprise pipetting or m icrofluidics.

I n the methods as taught herein, the cells may be contacted with the photoresponsive organic particles in such a manner that the photoresponsive organic particles can interact with - such as bind - the cells.

I n embodiments of the methods as taught herein, the cells and the one or more photoresponsive particles are contacted, and the mixture of the cells and the one or more photoresponsive organic particles is irradiated before the m ixture of the cells and the one or more photoresponsive organic particles is contacted with the cargo (i.e. before the cargo is added to the mixture of the cells and the one or more photoresponsive organic particles) .

I n embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles, wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; irradiating the m ixture of the cell and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell; and contacting the m ixture of the cell and the one or more photoresponsive organic particles with a cargo (e.g. adding the cargo to the mixture of the cell and the one or more photoresponsive organic particles) , thereby delivering the cargo into the cell.

I n embodiments of the methods as taught herein, the cells, the one or more photoresponsive particles, and the cargo are contacted with each other before the m ixture of the cells, the one or more photoresponsive organic particles, and the cargo is irradiated.

I n embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles and a cargo, wherein the cargo is not bound to the one or more photoresponsive organic particles, and wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a mixture of the cell, the cargo, and the one or more photoresponsive organic particles; and irradiating the m ixture of the cell, the cargo, and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell; characterized in that the one or more photoresponsive organic particles have a particle size of between 100 nm and 550 nm , more preferably between 150 and 550 nm , more preferably between 100 and 500 nm , more preferably between 200 and 400 nm , more preferably 200 and 300 nm , more preferably of between 225 and 275 nm , more preferably between 230 and 260 nm , more preferably between 240 and 255 nm , such as 250 nm or wherein said particle size is between 150 and 550 nm , more preferably between 200 and 550 nm , more preferably between 300 and 550 nm , more preferably 400 and 550 nm , more preferably of between 450 and 550 nm , such as 500 nm ..

I n embodiments, the cells, the one or more photoresponsive particles, and the cargo may be contacted with each other in any order. I n embodiments, the cargo may be contacted with the cells before contacting the mixture of the cells and the cargo with the photoresponsive organic particles. I n preferred embodiments, the cargo may be contacted with the cells after contacting the cells with the photoresponsive organic particles. Further, in embodiments, the cargo may be contacted with the photoresponsive organic particles before contacting the m ixture of the photoresponsive organic particles and the cargo with the cells.

Accordingly, in preferred embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles, wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, and a combination thereof, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; contacting the m ixture of the cell and the one or more photoresponsive organic particles with the cargo (e.g. adding the cargo to the m ixture of the cell and the one or more photoresponsive organic particles) ; and irradiating the m ixture of the cell, the cargo, and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell. I n embodiments, the methods as taught herein may comprise: contacting the cargo with the cell (e.g. adding the cargo to a cell suspension) , thereby obtaining a m ixture of the cell and the cargo; contacting the m ixture with one or more photoresponsive organic particles, wherein the organic particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle , and a combination thereof; and irradiating the m ixture with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell. I n embodiments, the methods as taught herein may comprise: contacting the cargo with one or more photoresponsive organic particles, wherein the cargo is not bound to the one or more photoresponsive organic particles, and wherein the organic particle is selected from the group consisting of a polymer- based particle, a protein-based particle, a lipid-based particle , and a combination thereof, thereby obtaining a m ixture of the cargo and the one or more photoresponsive organic particles; contacting the m ixture with a cell; and irradiating the m ixture with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell.

I n embodiments, the methods as taught herein may comprise contacting the cell with the one or more photoresponsive organic particles, thereby allowing (at least part of) the one or more photoresponsive organic particles to become associated to the cells, e.g. by binding to the cell membrane or cell wall or by active internalization by endocytic processes. I n embodiments, the methods as taught herein may comprise washing the unbound photoresponsive organic particles from the cells.

I n embodiments, the methods as taught herein may comprise contacting a cell with one or more photoresponsive organic particles, thereby obtaining a m ixture of the cell and the one or more photoresponsive organic particles; and optionally washing the unbound photoresponsive organic particles from the cells.

I n embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; optionally washing the unbound photoresponsive organic particles from the cells; contacting the m ixture of the cell and the one or more photoresponsive organic particles with the cargo (e.g. adding the cargo to the m ixture of the cell and the one or more photoresponsive organic particles) ; and irradiating the m ixture of the cell, the cargo, and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell.

I n embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; optionally washing the unbound photoresponsive organic particles from the cells; irradiating the m ixture of the cell and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell; and contacting the m ixture of the cell and the one or more photoresponsive organic particles with the cargo (e.g. adding the cargo to the mixture of the cell and the one or more photoresponsive organic particles) , thereby delivering the cargo into the cell.

I n embodiments, the methods as taught herein may comprise incubating the mixture of the cells and the photoresponsive organic particles. I ncubation may increase the binding of the one or more photoresponsive organic particles to the cells.

I n embodiments, the methods as taught herein may comprise contacting a cell with one or more photoresponsive organic particles, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; and optionally incubating the m ixture of the cells and the photoresponsive organic particles.

I n embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; incubating the m ixture of the cells and the photoresponsive organic particles; optionally washing the unbound photoresponsive organic particles from the cells; contacting the m ixture of the cell and the one or more photoresponsive organic particles with the cargo (e.g. adding the cargo to the mixture of the cell and the one or more photoresponsive organic particles) ; and irradiating the m ixture of the cell, the cargo, and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell.

I n embodiments, the methods as taught herein may comprise: contacting a cell with one or more photoresponsive organic particles, thereby obtaining a mixture of the cell and the one or more photoresponsive organic particles; incubating the m ixture of the cells and the photoresponsive organic particles; optionally washing the unbound photoresponsive organic particles from the cells; irradiating the m ixture of the cell and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell; and contacting the m ixture of the cell and the one or more photoresponsive organic particles with the cargo (e.g. adding the cargo to the mixture of the cell and the one or more photoresponsive organic particles) , thereby delivering the cargo into the cell.

I n embodiments of the methods as taught herein, the cells and the photoresponsive organic particles may be contacted immediately before perform ing the next step. This advantageously allows to perform rapid delivery of a cargo into cells. I n embodiments, after contacting (e.g. m ixing) the cells and the photoresponsive organic particles, the cells and the photoresponsive organic particles may be incubated at least 1 m in. I n embodiments, after contacting (e.g. m ixing) the cells and the photoresponsive organic particles, the cells and the photoresponsive organic particles may be incubated at least 5 m in, such as at least 10 min, at least 15 m in, at least 30 min, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours.

I n embodiments of the methods as taught herein, after contacting (e.g. m ixing) the cells and the photoresponsive organic particles, the cells and the photoresponsive organic particles may be incubated before (i.e. prior to) contacting the cargo with the mixture of the cells and the photoresponsive organic particles. I n embodiments, after contacting (e.g. mixing) the cells and the photoresponsive organic particles, the cells and the photoresponsive organic particles may be incubated at least 1 min before (i.e. prior to) contacting the cargo with the m ixture of the cells and the photoresponsive organic particles. I n embodiments, after contacting (e.g. m ixing) the cells and the photoresponsive organic particles, the cells and the photoresponsive organic particles may be incubated at least 5 min, such as at least 10 m in, at least 15 min, at least 30 min, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours before (i.e. prior to) contacting the cargo with the mixture of the cells and the photoresponsive organic particles.

I n embodiments, the methods as taught herein may comprise contacting the cell with a photoresponsive organic particle and the cargo in an aqueous solution, such as in a cell culture medium , commonly a liquid cell culture medium . Typically, the cell culture medium will comprise a basal medium form ulation as known in the art. Many basal media form ulations (available, e.g., from the American Type Culture Collection, ATCC; or from Thermo Fisher Scientific, Waltham , Massachusetts, USA) can be used to culture the cells herein, including but not lim ited to Eagle's Minimum Essential Medium (MEM) , Dulbecco’s Modified Eagle’s Medium (DMEM) , alpha modified Minimum Essential Medium (alpha-MEM) , Basal Medium Essential (BME) , BGJb, F-12 Nutrient Mixture (Ham) , Iscove’s Modified Dulbecco’s Medium (I MDM) , or Opti-MEM (reduced serum medium) , available from Thermo Fisher Scientific (Waltham , Massachusetts, USA) , and modifications and/or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured. Such basal media formulations contain ingredients necessary for cell development, which are known per se. By means of illustration and not lim itation, these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn) , physiological buffers (e.g. , HEPES, bicarbonate) , nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, am ino acids, vitam ins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, sodium pyruvate, sodium acetate) , etc.

I n embodiments, the methods as taught herein may comprise contacting the cell

5 with a photoresponsive organic particle and the cargo in an aqueous solution, such as a suitable transfection buffer solution, such as Dulbecco’s Phosphate Buffered Saline (DPBS) .

I n embodiments, the methods as taught herein may comprise the prior step of providing the cells, the cargo, and the photoresponsive organic particles. Each of the cells, the cargo and the photoresponsive organic particles may be provided in an aqueous solution as taught herein.

I n embodiments, the methods as taught herein may comprise providing the cells. I n embodiments, the methods as taught herein may comprise providing the cells in an5 aqueous solution, such as in a cell culture medium , com monly a liquid cell culture medium .

I n embodiments, the methods as taught herein may comprise providing the cargo. I n embodiments, the methods as taught herein may comprise providing the cargo in0 an aqueous solution, such as in water.

I n embodiments, the methods as taught herein may comprise providing the photoresponsive organic particles. I n embodiments, the methods as taught herein may comprise providing the photoresponsive organic particles in an aqueous5 solution, such as in a cell culture medium , com monly a liquid cell culture medium .

I n embodiments, the methods as taught herein may comprise providing the cells as adherent cells. I n embodiments, the methods as taught herein may comprise contacting adherent cells with one or more photoresponsive organic particles. I n embodiments, the methods as taught herein may comprise contacting adherent cells0 with one or more photoresponsive organic particles and the cargo.

I n embodiments, the methods as taught herein may comprise providing the cells in suspension. I n embodiments, the methods as taught herein may comprise contacting the cells in suspension with a photoresponsive organic particle and the cargo. 5 The terms “suspension” and “cell suspension” generally refer to a heterogenous mixture comprising cells dispersed in a liquid phase. As the mixture is generally liquid, the cells may in principle be able to, but need not, settle or sediment from the mixture. Cells such as animal cells including human cells may be “adherent”, i.e. , require a surface for growth, and typically grow as an adherent monolayer on said surface (i.e., adherent cell culture) , rather than as free-floating cells in a culture medium (suspension culture) . Adhesion of cells to a surface, such as the surface of a tissue culture plastic vessel, can be readily examined by visual inspection under inverted m icroscope. Cells grown in adherent culture require periodic passaging, wherein the cells may be removed from the surface enzymatically (e.g., using trypsin) , suspended in growth medium , and re-plated into new culture vessel(s) . I n general, a surface or substrate which allows adherence of cells thereto may be any substantially hydrophilic substrate. As known in the art, tissue culture vessels, e.g., culture flasks, well plates, dishes, or the like, may be usually made of a large variety of polymeric materials, suitably surface treated or coated after moulding in order to provide for hydrophilic substrate surfaces.

I n embodiments, the methods as taught herein may thus comprise the prior step of suspending the cells in an aqueous solution, such as a cell culture medium . I n embodiments, adherent cells may be first removed from a surface by a method as known in the art, for instance by tapping the cell culture vessel, by scraping the surface, or enzymatically (e.g., using trypsin or Accutase® , followed by washing) . I n embodiments of the methods as taught herein, the method may be performed: in an aqueous solution ; at physiological conditions; at a temperature ranging from 15°C to 40°C, preferably at a temperature ranging from 20°C to 25°C or from 32°C to 37°C; and/or at a pH ranging from about 3 to about 1 1 , preferably from about 5 to about 7.

I n embodiments, the methods as taught herein may comprise irradiating the mixture of the cell and the one or more photoresponsive organic particles with electromagnetic radiation, thereby causing permeabilization of a barrier of the cell. I n embodiments, the methods as taught herein may comprise irradiating the mixture of the cell, the cargo, and the photoresponsive organic particle with electromagnetic radiation.

By irradiating the m ixture, the photoresponsive organic particles capable of absorbing electromagnetic radiation may be optically activated. Upon absorption of the electromagnetic radiation by the photoresponsive organic particles, photothermal or photochem ical effects can be induced. Of particular interest is the generation of a thermal or plasma induced vapour bubble, for example a vapour m icrobubble or a vapour nanobubble. By the generation of a vapour bubble, local high-pressure waves may be induced which may alter a nearby cell, for example a cellular barrier of a nearby cell.

The phrase “generation of a vapour bubble” includes expansion of the vapour bubble, collapse of the vapour bubble, or a combination of expansion and collapse of the vapour bubble, and secondary effects that can be the result of the bubble expansion and collapse, such as pressure waves and flow of the surrounding medium . The terms “vapour bubble” or “bubble” as used herein refer to vapour nanobubbles and vapour microbubbles. Preferably, a vapour bubble may have a diameter in the range of 10 nm to 100 pm. Vapour bubbles may comprise water vapour bubbles.

The phrases “cellular barrier” or “barrier of the cell” refer to cell membranes (plasma membranes) or cell walls of eukaryotic and prokaryotic cells, and intracellular membranes, such as endosomal membranes, nuclear envelopes, mitochondrial membranes.

The terms “alter”, “altering” or “alteration” refer to any way to change one or more properties of a cell, for example at least locally, such as a barrier of a cell. Altering includes but is not lim ited to inducing a local change in a cell’s composition, for example in the composition of a barrier of the cell by adding, removing, destroying or reorganizing constituents. Altering comprises for example changing one or more physicochem ical properties, such as its viscosity, porosity, density, rigidity, elasticity etc. Altering also includes local destruction or rearrangement of cellular barrier constituents, resulting in a change of the composition and/or physicochemical properties of the cellular barrier. Altering includes amongst other deforming, permeabilizing and perforating.

The terms “deform”, “deforming” and “deformation” refer to any way to alter the spatial organization or structure of a cell, in particular of the cellular barrier, at least partially, for example at least locally. Examples of deforming comprise providing a cellular barrier with indentations or invaginations.

The terms “permeabilize”, “permeabilizing” and “permeabilization” refer to any way to alter the permeability of a cell, in particular of the permeability of the cellular barrier, at least partially, for example at least locally. Examples of permeabilizing comprise altering the barrier composition or structure so that it becomes more permeable to a cargo. The terms “perforate”, “perforating” or “perforation” refer to any way to provide a cell, in particular the cellular barrier, at least partially, for example at least locally, with one or more openings, holes or pores. By perforating a cellular barrier, openings are created into the barrier allowing the transport of a cargo across or into that barrier.

The terms “perforate”, “perforating”, “perforation” and the terms “increase the permeability of”, “permeabilize”, “permeabilizing”, “permeabilization” are interchangeably used. Similarly, the terms “opening”, “hole” and “pore” may be used interchangeably herein.

The m ixture is preferably irradiated by a laser such as a pulsed radiation source, although irradiation by a continuous wave radiation source can also be considered.

The m ixture can be irradiated by one or more pulses.

The terms “radiation” and “electromagnetic radiation” may be used interchangeably herein.

The wavelength of the radiation source may range from the ultraviolet region to the infrared region. I n preferred methods, the wavelength range of the radiation used is in the visible to the infrared region, including the near infrared region.

I n embodiments of the methods as taught herein, the electromagnetic radiation may be generated by a laser, such as a pulsed laser. Laser irradiation, such as irradiation by pulsed lasers, e.g. pico-, femto- and/or nanosecond pulsed lasers, can be combined with a photoresponsive organic particle in accordance with embodiments of the present invention to efficiently permeabilize a cell’s barrier, e.g. by laser- induced vapour nanobubble generation. While laser irradiation may be advantageous, irradiation by another (intense) light source is not necessarily excluded, e.g. a Xenon flash lamp, to achieve the same or sim ilar effects.

The laser pulses may each have a power density or intensity in the range of 10 ⁴ to 10 ¹⁷ W/cm2, e.g. in the range of 10 ⁶ to 10 ¹⁵ W/cm2, 10 ⁷ to 10 ¹⁴ W/cm2, or 10 ⁸ to 10 ¹³ W/cm2.

The laser pulses may each have a fluence (electromagnetic energy delivered per unit area) in the range of 0.01 J/ cm ² to 100 J/cm ² , 0.05 J/ cm ² to 50 J/ cm ² , 0.1 J/ cm ² to 10 J/cm ² , or 0.5 J/cm ² to 10 J/cm ² , e.g. in the range of 1 J/cm ² to 10 J/cm ² . The laser pulses may consist of 1 to 1000 laser pulses, such as 1 to 500 laser pulses, 1 to 100 laser pulses, 1 to 20 laser pulses, or 1 to 10 laser pulses (per cell or per cell sample) . The number of laser pulses may be depending on the photoresponsive organic particle, the type of cargo, and the type of cells.

The laser pulses may have a duration in the range of 1 fs to 100 seconds (s) , for instance in the range of 100 fs to 1 s, e.g. in the range of 1 ps to 0.1 s or in the range of 1 ns to 100 ps.

I n embodiments of the methods as taught herein, the electromagnetic radiation may be generated by a laser, such as a pulsed laser, wherein: the intensity of the pulses of the laser may be at least 104 W/cm2, such as 104 to 1017 W/cm2; the fluence of the pulses of the laser may be at least 0.01 mJ/ cm ² , such as 0.01 J/cm ² to 100 J/ cm ² ; the number of pulses of the laser may be at least 1 laser pulse; such as 1 to 1000 laser pulses; and/or the duration of the pulses of the laser may be at least 1 fs, such as 1 fs to 100 s.

I n embodiments of the methods as taught herein, the electromagnetic radiation may be generated by a laser, such as a pulsed laser, wherein the intensity of the pulses of the laser may be 10 ⁸ to 10 ¹³ W/cm2; the fluence of the pulses of the laser may be 1 J/cm ² to 10 J/cm ² ; the number of pulses of the laser may be 1 to 10 laser pulses; and/or the duration of the pulses of the laser may be 1 ns to 100 ps.

I n embodiments of the methods as taught herein, the cells, the photoresponsive organic particles, and optionally the cargo, may be contacted immediately before irradiating the m ixture of the cells, the photoresponsive organic particles, and optionally the cargo. This advantageously allows to perform rapid delivery of a cargo into cells.

I n embodiments of the methods as taught herein, after contacting (e.g. m ixing) the cells, the photoresponsive organic particles, and optionally the cargo, the m ixture of the cells, the photoresponsive organic particles, and optionally the cargo, may be incubated before (i.e. prior to) irradiating the m ixture. I n embodiments, after contacting (e.g. m ixing) the cells, the photoresponsive organic particles, and optionally the cargo, the m ixture of the cells, the photoresponsive organic particles, and optionally the cargo, may be incubated at least 1 m in before (i.e. prior to) irradiating the m ixture. I n embodiments, after contacting (e.g. m ixing) the cells, the photoresponsive organic particles, and optionally the cargo, the m ixture of the cells, the photoresponsive organic particles, and optionally the cargo, may be incubated at least 5 min, such as at least 10 m in, at least 15 min, at least 30 min, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours before (i.e. prior to) irradiating the m ixture.

I n embodiments, the m ixture of the cells and the one or more photoresponsive organic particles is contacted with the cargo (i.e. the cargo is added to the m ixture) immediately before irradiating (e.g. applying laser irradiation to) the m ixture. I n embodiments, the mixture of the cells and the one or more photoresponsive organic particles is contacted with the cargo (i.e. the cargo is added to the mixture) at most 15 min, at most 10 m in, at most 5 min , at most 2 m in, at most 1 min, at most 30 seconds, or at most 10 seconds before irradiating the mixture. Thereby, the cargo is present in the mixture to enter into the cells upon permeabilization of a barrier of the cell.

I n embodiments, the m ixture of the cells and the one or more photoresponsive organic particles is contacted with the cargo (i.e. the cargo is added to the m ixture) im mediately after irradiating the mixture. I n embodiments, the m ixture of the cells and the one or more photoresponsive organic particles is contacted with the cargo (i.e. the cargo is added to the mixture) at most 15 m in, at most 10 m in, at most 5 m in , at most 2 min, at most 1 m in, at most 30 seconds, or at most 10 seconds after irradiating the m ixture. Thereby, the permeabilized cells allow entry of the cargo into the cells before the pores that are created in the cells will be closed again.

The methods as taught herein may be suitable to alter, manipulate and/or treat cells. I n embodiments, the methods may be in particular suitable to perforate or permeabilize cells, in particular cellular barriers. By the methods as taught herein, transient pores can be formed into a cellular barrier, such as a cell membrane or cell wall, allowing the delivering of a cargo in the cells. I n embodiments, the methods as taught herein may be in particular suitable for use in drug delivery, in intracellular delivery of cargo, in cell therapy, in im munotherapy, in gene therapy and in transfection of cells for example stem cells or T cells.

I n embodiments, the methods as taught herein may be suitable for use in intracellular delivery of nucleic acids, including oligonucleotides, siRNA, m RNA or pDNA. I n embodiments, the methods as taught herein may be also suitable for use in the intracellular delivery of nucleoproteins, including ribonucleoproteins, such as Cas9/gRNA. Furthermore, the methods as taught herein may be suitable for use in the intracellular delivery of peptides and proteins, such as nanobodies or antibodies. I n addition, the methods may be suitable for use in the intracellular delivery of polysaccharides, such as labelled polysaccharides allowing detection of the cells. The methods as taught herein furthermore allow to alter, treat and/or manipulate cells with high throughput.

The methods as taught herein may as well be useful for the delivery of a cargo into a cell of a human or animal body.

A further aspect provides a photoresponsive organic particle as defined herein and a cargo as defined herein, for use in an in vivo method of delivering a cargo into a cell of a subject. For instance, photoresponsive organic particles and a cargo may be administered in vivo to a subject, for instance in the proxim ity of cells of the subject, e.g. by injection into a tissue such as subcutaneous injection, and the cells may be irradiated with electromagnetic radiation, thereby causing permeabilization of a barrier of the cells and delivering the cargo into the cells. The cargo may be a therapeutic agent. The method may advantageously be a method of treatment.

Hence, a further aspect provides a photoresponsive organic particle as defined herein and a cargo as defined herein, for use in a method of therapy or treatment of a disease in a subject, wherein a cargo is delivered to a cell of a subject.

An aspect provides one or more photoresponsive organic particles as defined herein and a cargo as defined herein, for use in an in vivo method of delivering a cargo into a cell of a subject, the method comprising: adm inistering the one or more photoresponsive organic particles to the surrounding (i.e. in the proxim ity) of the cell of the subject; irradiating at least part of the surrounding of the cell of the subject, thereby causing permeabilization of a barrier of the cell; and adm inistering the cargo to the surrounding of the cell of the subject, thereby delivering the cargo into the cell; or the method comprising: adm inistering the one or more photoresponsive organic particles and the cargo to the surrounding of the cell of the subject; and irradiating at least part of the surrounding of the cell of the subject, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell. A further aspect relates to one or more photoresponsive organic particles as defined herein and a cargo as defined herein, for use in a method of therapy or treatment of a disease in a subject, wherein a cargo is delivered to a cell of a subject, the method comprising: adm inistering the one or more photoresponsive organic particles to the surrounding (i.e. in the proxim ity) of the cell of the subject; irradiating at least part of the surrounding of the cell of the subject, thereby causing permeabilization of a barrier of the cell; and adm inistering the cargo to the surrounding of the cell of the subject, thereby delivering the cargo into the cell; or the method comprising: adm inistering the one or more photoresponsive organic particle and the cargo to the surrounding of the cell of the subject; and irradiating at least part of the surrounding of the cell of the subject, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell. Related aspects provide: a method of treatment of a disease in a subject, wherein a cargo is delivered in vivo to a cell of a subject in need of such a treatment, the method comprising adm inistering a therapeutically effective amount of one or more photoresponsive organic particles as defined herein and a cargo as defined herein to the subject. the use of one or more photoresponsive organic particles as defined herein and a cargo as defined herein for the manufacture of a medicament for therapy or treatment of a disease, wherein a cargo is delivered to a cell of a subject. the use of one or more photoresponsive organic particles as defined herein and a cargo as defined herein for therapy or treatment of a disease in a subject, wherein a cargo is delivered in vivo to a cell of a subject.

I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be adm inistered separately. I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be dosed independently, i.e. are present in a kit of parts in different unit doses or dosage forms. Said separate dosage forms can be administered sim ultaneously and/or at different time points, such as chronologically staggered, that is at different time points. The photoresponsive organic particle as taught herein and the cargo as taught herein can be administered by the same route or by different routes.

I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be administered sequentially. I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be adm inistered separately and sequentially. I n embodiments, the photoresponsive organic particles may be adm inistered before the cargo such as the therapeutic agent. I n embodiments, the photoresponsive organic particles may be administered immediately before the cargo such as the therapeutic agent.

I n embodiments, the photoresponsive organic particles may be adm inistered to the surrounding of the cells at least 1 min before adm inistration of the cargo, such as the therapeutic agent. I n embodiments, the photoresponsive organic particles may be adm inistered to the surrounding o the cell at least 5 min, such as at least 10 m in, at least 15 min, at least 30 min, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours before adm inistration of the cargo, such as the therapeutic agent.

I n embodiments, the irradiation step is performed after the administration of the one or more photoresponsive organic particles. I n embodiments, the irradiation step is performed before or after the administration of the cargo.

I n embodiments of the methods or uses as taught herein, the method may comprise: administering the one or more photoresponsive organic particle to the surrounding (i.e. in the proxim ity) of the cell of the subject; irradiating at least part of the surrounding of the cell of the subject, thereby causing permeabilization of a barrier of the cell; and administering the cargo to the surrounding of the cell of the subject, thereby delivering the cargo into the cell;

I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be adm inistered simultaneously. I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be administered separately and simultaneously.

I n embodiments of the methods or uses as taught herein, the method may comprise: administering the one or more photoresponsive organic particles and the cargo to the surrounding of the cell of the subject; and irradiating at least part of the surrounding of the cell of the subject, thereby causing permeabilization of a barrier of the cell and delivering the cargo into the cell. I n embodiments, the photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent may be comprised in a composition for sim ultaneous adm inistration or in a kit of parts for sim ultaneous or sequential adm inistration. As used herein, the phrases “administering the one or more photoresponsive organic particles to the surrounding of the cells” or “administering the one or more photoresponsive organic particles in the proxim ity of the cells” refer to adm inistering the photoresponsive organic particles within a distance of the cell that allow inducing permeabilization of a barrier of the cell after irradiation with electromagnetic radiation.

I n certain embodiments of the methods as taught herein, the distance between the photoresponsive organic particle and the cell may be at most 10 pm, such as from about 0 pm to about 10 pm. For instance, the distance between the photoresponsive organic particle and the cell may be at most 1 pm, at most 100 nm, at most 10 nm, or at most 1 nm .

I n embodiments of the photoresponsive organic particle and cargo for use as taught herein : the cell may be an animal cell; the cell may be a human cell; and/or the cell may be an im mune cell; preferably wherein the imm une cell is a T cell, a lymphocyte, a macrophage, a dendritic cell, a monocyte, a NK cell, a NKT cell, a B cell, a neutrophil, a granulocyte, a microglial cell, or a Langerhans cell; preferably wherein the T cell is a naive T cell.

I n certain embodiments of the methods as taught herein, a cell population wherein said cell population has been transfected with the photoresponsive organic particle and a cargo, wherein after transfection at least 50% of the cell population has received the cargo, wherein the delivery efficiency of said cargo is at least 80% ; wherein said photoresponsive organic particle is polydopamine; wherein said polydopamine diameter is of about 100 nm to about 550 nm ; preferably between 150 and 550 nm , more preferably between 100 and 500 nm , more preferably between 200 and 400 nm , more preferably 200 and 300 nm , more preferably of between 225 and 275 nm , more preferably between 230 and 260 nm , more preferably between 240 and 255 nm , such as 250 nm or wherein said particle size is between 150 and 550 nm , more preferably between 200 and 550 nm , more preferably between 300 and 550 nm , more preferably 400 and 550 nm , more preferably of between 450 and 550 nm , such as 500 nm .. I n embodiments of the methods as taught herein, the diameter of polydopam ine particles measured by using any standard techniques that is known to the skilled person in the art, wherein said standard techniques are preferably Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) . I n certain embodiments of the photoresponsive organic particle and cargo for use as taught herein : the cargo is a macromolecule; preferably wherein the macromolecule is RNA; preferably said RNA is m RNA.

I n some embodiments “percentage of the cells that received the cargo” and “delivery yield” can be used interchangeably wherein the percentage of the cells that received the cargo calculated same as the percentage of delivery yield.

The term “delivery yield” as used herein refers to the ratio of the quantity of living (viable) cells comprising the cargo after perform ing the method as taught herein (e.g. the quantity of living cells comprising the cargo as detected after the delivery method) relative to the quantity of living (viable) cells before perform ing the method as taught herein (e.g. the quantity of living cells as detected before the delivery method) .

I n embodiments of the photoresponsive organic particle and cargo for use as taught herein, the electromagnetic radiation is generated by a laser, such as a pulsed laser. I n embodiments, the treatment may comprise perform ing laser irradiation of the photoresponsive organic particles as taught herein, in particular pulsed laser irradiation of the photoresponsive organic particles as taught herein. Accordingly, in embodiments, the treatment as taught herein comprises laser-assisted treatment.

I n embodiments of the photoresponsive organic particle and cargo for use as taught herein, administering the photoresponsive organic particle and the cargo to the surrounding of the cell may comprise injection, such as subcutaneous injection or intravascular injection. I n embodiments, administering the photoresponsive organic particle and the cargo to the surrounding of the cell may comprise intratumoral injection. I njection allows delivery of the photoresponsive organic particles and/or the cargo such as the therapeutic agent directly to the surrounding of the cells by a minimally invasive technique, thereby reducing the risks and pain for the patient and increasing the patient’s well-being.

I n embodiments, the in vivo methods may be useful for vaccination purpose. For instance, cells may be transfected in vivo with a cargo, such as m RNA and/or proteins, for vaccination purposes. The photoresponsive organic particles as taught herein and the cargo as taught herein such as the therapeutic agent allow treatment, such as laser-assisted treatment, of a disease or condition in a subject.

The terms “subject”, “individual” or “patient” can be used interchangeably herein, and typically and preferably denote humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g., non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like. The term “non-human animals” includes all vertebrates, e.g., mam mals, such as non-human primates, (particularly higher primates) , sheep, dog, rodent (e.g. mouse or rat) , guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. I n certain embodiments, the subject is a non-human mam mal. I n certain embodiments, the subject is a human subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and m ice. The term subject is further intended to include transgenic species.

Suitable subjects may include without lim itation subjects presenting to a physician for a screening for a disease or condition, subjects presenting to a physician with symptoms and signs indicative of a disease or condition, subjects diagnosed with a disease condition, and subjects who have received an alternative (unsuccessful) treatment for a disease or condition.

As used herein, a phrase such as “a subject in need of treatment” includes subjects that would benefit from treatment of a given condition. Such subjects may include, without lim itation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in who said condition is to be prevented.

The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed disease or condition, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of a disease or condition. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration of the disease state, and the like. The term may encompass ex vivo or in vivo treatments. The uses and methods as taught herein allow to administer a therapeutically effective amount of the active compound, such as the photoresponsive organic particle as taught herein and/or the cargo as taught herein, in subjects having a disease which

5 will benefit from such treatment. The term “therapeutically effective amount” as used herein, refers to an amount of active compound that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated.

The term “therapeutically effective dose” refers to an amount of an active compound, such as the photoresponsive organic particle as taught herein and/or the cargo as taught herein, that when administered brings about a positive therapeutic response with respect to treatment of a patient having the disease or condition being treated. 5 Appropriate therapeutically effective doses of an active compound, such as the photoresponsive organic particle as taught herein and/or the cargo as taught herein, may be determined by a qualified physician with due regard to the nature of the agent, the disease condition and severity, and the age, size and condition of the patient. 0

I n certain embodiments, the active compound, such as the photoresponsive organic particle as taught herein and/or the cargo as taught herein, e.g. the therapeutic agent, may be formulated into and administered as pharmaceutical form ulations or pharmaceutical compositions. 5

I n certain embodiments, the active compound, such as the photoresponsive organic particle as taught herein and/or the cargo as taught herein, e.g. the therapeutic agent, may be form ulated into a kit of parts and administered sim ultaneously or sequentially. 0

I n embodiments, the photoresponsive organic particles may be comprised in a pharmaceutical form ulation. I n embodiments, the cargo such as the therapeutic agent may be comprised in a pharmaceutical form ulation. 5 I n embodiments, the photoresponsive organic particle and the cargo such as the therapeutic agent may be comprised in a pharmaceutical formulation. The photoresponsive organic particles or pharmaceutically acceptable salts thereof, and/or the cargo such as the therapeutic agent or pharmaceutically acceptable salts thereof can be form ulated as an aqueous solution.

Accordingly, an aspect relates to a pharmaceutical formulation comprising a photoresponsive organic particles as taught herein. A further aspect provides a pharmaceutical form ulation comprising a photoresponsive organic particles as taught herein and a cargo as taught herein such as a therapeutic agent.

A further aspect relates to a pharmaceutical form ulation as taught herein for use in a method of therapy or treatment in a subject. Preferably, the subject is a human subject.

The terms “pharmaceutical composition”, “pharmaceutical form ulation” or “pharmaceutical preparation” may be used interchangeably herein and refer to a m ixture comprising an active ingredient. The terms “composition” or “formulation” may likewise be used interchangeably herein.

The terms “active ingredient”, “active compound” or “active component” can be used interchangeably and broadly refer to a compound or substance which, when provided in an effective amount, achieves a desired outcome. The desired outcome may be therapeutic and/or prophylactic. Typically, an active ingredient may achieve such outcome(s) through interacting with and/or modulating living cells or organisms.

The term “active” in the recitations “active ingredient” or “active component” refers to “pharmacologically active” and/or “physically active”.

I n embodiments, the pharmaceutical form ulations as taught herein may comprise in addition to the photoresponsive organic particles and the cargo such as the therapeutic agent one or more pharmaceutically acceptable excipients.

The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

As used herein, “carrier” or “excipient” includes any and all solvents, diluents, buffers (such as, e.g. , neutral buffered saline or phosphate buffered saline) , solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione) , am ino acids (such as, e.g., glycine) , proteins, disintegrants, binders, lubricants, wetting agents, em ulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.

Pharmaceutical compositions as intended herein may be form ulated for essentially any route of administration, such as without limitation, oral adm inistration (such as, e.g. , oral ingestion) , parenteral adm inistration (such as, e.g., subcutaneous, intravenous or intramuscular injection or infusion) , and the like.

For example, for oral administration, pharmaceutical compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g. , sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions. I n an example, without limitation, preparation of oral dosage forms may be is suitably accomplished by uniformly and intimately blending together a suitable amount of the active compound in the form of a powder, optionally also including finely divided one or m ore solid carrier, and form ulating the blend in a pill, tablet or a capsule. Exemplary but non-lim iting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (such as, e.g., glucose, mannose, lactose or sucrose) , sugar alcohols (such as, e.g. , mannitol) , dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Compressed tablets containing the pharmaceutical composition can be prepared by uniformly and intimately mixing the active ingredient with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the m ixture in a suitable machine to the shape and size desired. Moulded tablets maybe made by moulding in a suitable machine, a m ixture of powdered compound moistened with an inert liquid diluent. Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, sem isolid and liquid polyols, natural or hardened oils, etc.

Preferably the pharmaceutical form ulation may be form ulated for parenteral adm inistration, e.g. by injection.

I n embodiments, the pharmaceutical composition may be form ulated as an aqueous solution. For example, for parenteral administration, pharmaceutical compositions may be advantageously form ulated as solutions, suspensions or em ulsions with suitable solvents, diluents, solubilisers or em ulsifiers, etc. Suitable solvents are, without lim itation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively m ixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1 ,3- butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The photoresponsive organic particles and/or the cargo or pharmaceutically acceptable salts thereof can also be lyophilized. The obtained lyophilizates can be used, for example, for injection or infusion preparation or for the production of injection or infusion preparations.

I n embodiments, the photoresponsive organic particles may be comprised in a kit of parts.

I n embodiments, the photoresponsive organic particles and the cargo such as the therapeutic agent may be comprised in a kit of parts.

A further aspect relates to a kit of parts comprising a photoresponsive organic particles as taught herein. A further aspect provides a kit of parts comprising a photoresponsive organic particles as taught herein and a cargo such as a therapeutic agent as taught herein.

A further aspect thus relates to a kit of parts as taught herein for use in a method of therapy or treatment in a subject. Preferably, the subject is a human subject.

The terms “kit of parts”, “kit-of-parts” or “kit” as used herein refer to a product containing components necessary for carrying out the specified uses or methods, packed so as to allow their transport and storage. Materials suitable for packing the components comprised in a kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate) , bottles, flasks, vials, ampules, paper, envelopes, or other types of containers, carriers or supports. Where a kit comprises a plurality of components, at least a subset of the components (e.g., two or more of the plurality of components) or all of the components may be physically separated, e.g. , comprised in or on separate containers, carriers or supports. The components comprised in a kit may be sufficient or may not be sufficient for carrying out the specified uses or methods, such that external reagents or substances may not be necessary or may be necessary for perform ing the methods, respectively. Typically, kits are employed in conjunction with standard laboratory equipment, such as liquid handling equipment, environment (e.g., temperature) controlling equipment, analytical instruments, etc. I n addition to the photoresponsive organic particles as taught herein and/or the cargo as taught herein such as the therapeutic agent, optionally provided on arrays or microarrays, the present kits may also include excipients such as solvents useful in the specified uses or methods. Typically, the kits may also include instructions for use thereof, such as on a printed insert or on a computer readable medium . The terms may be used interchangeably with the term “article of manufacture”, which broadly encompasses any man-made tangible structural product, when used in the present context.

I n a final aspect the current disclosure is also directed to a cell or cell population wherein said cell or cell population is obtainable by transfection with a method according to any of the foregoing. I n an embodiment, and after transfection at least 50% , more preferably 55% , more preferably 60% , more preferably 65% , more preferably 70% , more preferably 75% , more preferably 80% , more preferably 85% , more preferably 90% , more preferably 95% up to 99% or even 100% of the cell population has received the cargo. I n another or further embodiment, said at least 50 to 99% of said cell population has received said cargo, more preferably at least 55% to 99% , more preferably at least 60% to 99% , more preferably at least 65% to 99% , more preferably at least 70% to 99% has received said cargo.

I n an embodiment, said delivery efficiency of said cargo is at least 80% , more preferably at least 85% , more preferably at least 90% , such as 91 % , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% to even 100% .

While any type of cargo as described above is possible, said cargo is preferably a nucleic acid, such as but not limited to small interfering RNA (siRNA) , m icro RNA (miRNA) , messenger RNA (m RNA) or plasm id DNA (pDNA) .

Any type of cells as described above can be used, but in a particularly preferred embodiment, said cells are T cells, more preferably naive T cells.

EXAMPLES and DESCRI PTI ON OF Fl GURES

Material and Methods

Synthesis of 1 00 nm , 250 nm and 500 nm polydopam ine nanopa ticles For 100 nm polydopam ine nanoparticles (hereafter PD NPs) , 250 mg of dopamine. HCI (Sigma-Aldrich) was dissolved into 100 m L HyClone water (VWR, HyPure, Cell Culture Grade) of 50 °C (2.5 mg/mL Dopamine. HCI) . Next, 1055 pL of a 1 M NaOH solution (molar ratio: 1 /0.8) was added to the dopamine solution under vigorous stirring. The m ixture was allowed to stir for about 1 hour. The solution was then collected and transferred to 1 .5 m L Eppendorf tubes. The 100 nm PD NPs were washed with HyClone water by centrifugation (21 .000 ref, 20 minutes) . To reduce agglomeration after centrifugation, tip sonication was applied for 30 seconds (10% A, Branson digital sonifier, Danbury, USA) .

For 250 nm PD NPs, 350 mg of dopamine. HCI (Sigma-Aldrich) was dissolved into 100 mL HyClone water (VWR, HyPure, Cell Culture Grade) of 50 °C (3.5 mg/mL Dopam ine. HCI) . Next, 1476 pL of a 1 M NaOH solution (molar ratio: 1 /0.8) was added to the dopamine solution under vigorous stirring. The m ixture was allowed to stir for about 2 hours. The solution was then collected and transferred to a 50 mL canonical tube. The 250 nm PD NPs were washed with HyClone water by centrifugation (4.000 ref, 20 m inutes) . To reduce agglomeration after centrifugation, tip sonication was applied for 30 seconds (10% A, Branson digital sonifier, Danbury, USA) .

For 500 nm PD NPs, 45 mg of dopamine. HCI (Sigma-Aldrich) was dissolved into 15 m L HyClone water (VWR, HyPure, Cell Culture Grade) of 50 °C (3 mg/m L Dopamine.HCI) . Next, 142 pL of a 1 M NaOH solution (molar ratio: 1 /0.6) was added to the dopamine solution under vigorous stirring. The mixture was allowed to stir for about 5 hours. The solution was then collected and transferred to a 50 m L canonical tube. The 500 nm PD NPs were washed with HyClone water by centrifugation (4.000 ref, 10 minutes) . To reduce the agglomeration by centrifugation, tip sonication was applied for 30 seconds (10% A, Branson digital sonifier, Danbury, USA) .

Functionalization of 1 00 nm , 250 nm and 500 nm polydopam ine nanoparticles w ith bovine serum album in

I n order to enhance their colloidal stability, the PD NPs were functionalized with bovine serum albumin (BSA, VWR Chemicals, Biotechnology grade, USA) . The uncoated PD NPs with different sizes were m ixed with 20 mg/mL BSA solution at a volume ratio of 1 : 1 . The mixture was then allowed to react by vigorous stirring overnight and the remaining unbound BSA was removed by several washing steps with HyClone water. The following centrifugation conditions were used: 20 min at 21 .000 ref for 100 nm PD-BSA NPs, 20 m in at 4000 ref for 250 nm PD-BSA NPs and 10 m in at 4000 ref for 500 nm PD-BSA NPs. The final dispersions of PD-BSA NPs were stored at 4 °C.

Polydopam ine m ediated photoporation in u nstim ulated hum an T cells

Human T cell cu ltu re

Primary CD3+ T cells were isolated from buffy coats of healthy donors, obtained after informed consent from the Red Cross Flanders (Red Cross, Ghent, Belgium) . Briefly, peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Lymphoprep (Stem Cell Technologies, Canada) , and subsequently used for CD3+ cell isolation by negative selection (EasySep, Stem Cell Technologies, Canada) according to the manufacturer’s protocol. Primary CD3+ T cells were kept in culture at 37 °C in a 5% CO2 atmosphere in I MDM Glutamax™(l nvitrogen, Belgium) , supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest) and 100 pg/mL penicillin/streptomycin (Gibco, Merelbeke, Belgium) . Cells were used for photoporation experiments on the day of isolation.

Polydopam ine m ediated photoporation for FI TC dextran 500 kDa delivery in unstim u lated hum an CD3+ T cells

2-fold dilutions of PD-BSA NP stock dispersions were prepared in Opti-MEM. Cells were first washed several times with Opti-MEM by centrifugation at 300 g for 4 m inutes. Next, 1 x10 ⁶ cells in 24 pL Opti-MEM were transferred per well of a flat bottom 96-well plate (VWR, plastic bottom) . 25 pL of PD NP dilutions at the desired concentrations were added to the cell suspension, followed by an addition of 1 pL of FITC labelled dextran 500 kDa (FD500, final concentration : 1 mg/mL) . For the untreated control (NTR) , Opti-MEM was added to the cells without PD-BSA NPs or FD500. Additionally, a control was included with cells incubated with Opti-MEM containing FD500 but without PD-BSA NPs. This was included to correct for any spontaneous uptake of FD500 by cells. Lastly, a “photoporation control” was performed where cells received laser irradiation after incubation with Opti-MEM and PD-BSA NPs but without FD500.

Cells were sedimented to the bottom of the plate by a short spin for 10 seconds at 500 g and immediately treated by pulsed laser illum ination with the desired energy levels. After phot oporation treatment, cells were supplemented with 150 pL complete culture medium . I n order to assess cell viability, a portion of the cell suspension (0.5x10 ⁵ cells) was transferred to a new plate prefilled with fresh culture medium and cells were allowed to rest in the incubator for at least 2 hours before perform ing the viability assay. The remaining cells were then washed three times by centrifugation (300 g, 5 m in) , resuspended in culture medium and incubated at 37 °C, 5% CO2 before analysis of transfection efficiency.

Flow cytom etry

Flow cytometry was used to evaluate the efficiency of FD500 delivery. Cells were washed with DPBS and resuspended in flow buffer composed of DPBS supplemented with 1 % bovine serum album in. TO-PRO-3 stain was added as an indicator of cell death. Samples were analyzed on a MACSQuant Analyzer 16 with a minimum of 50000 cells recorded per sample. Flow cytometry data were then analyzed using FlowJo software (Treestar I nc., USA) .

Cell viability assay

The viability of primary human T cells was assessed two hours after photoporation treatment using a CellTiter-Glo® lum inescent cell viability assay (Promega, Leiden, The Netherlands) , according to the manufacturer’s protocol. Briefly, 100 pL of cell suspension (0.5x10 ⁵ cells) was m ixed with an equal volume of CellTiter-Glo® reagent. The content of the plate was mixed for 10 m in using an orbital shaker (100 rpm) , after which 100 pL of the mixture was transferred to an opaque 96-well plate. The lum inescent signal was measured using a GloMax™ 96 lum inometer (Promega, Leiden, The Netherlands) . Cell viability for the different conditions was calculated relatively to the untreated samples.

Resu lts

The present invention is in no way lim ited to the embodiments described in the examples and/or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention. The hydrodynamic diameter of the different PD-BSA NPs was measured via Dynamic Light Scattering (DLS) . FD500 and eGFP-m RNA were delivered in unstimulated T cells by photoporation

Surprisingly, we showed that the delivery efficiency in naive T cells can be increased by using PD NPs with a size between 100 nm and 550 nm with the most optimal size depending on the type of cargo used (Figure 4) .