Technical Field of the Invention

The present invention relates to protein-based nanoparticles, synthesis of these nanoparticles and their use in cancer therapy for improving the stabilization and cellular uptake of MS1 peptide that specifically binds to and inhibits Mcl-1 protein.

State of the Art

It has been known for years that cancer is an important health problem for humanity. In 2020, approximately 19.3 million people were diagnosed with cancer and 10 million people died due to cancer. Activation of apoptosis pathways is an important mechanism in killing cancer cells. The increased expression of antiapoptotic proteins plays an important role in the formation of many cancers and especially in the resistance of cancerous cells to chemotherapy. The importance of studies on the inhibition of such antiapoptotic proteins in the field of cancer is increasing day by day.

Mcl-1 protein, one of the antiapoptotic proteins, is an antiapoptotic member of the BCL-2 family of proteins classified according to their function into three groups: antiapoptotic, proapoptotic and BH3-only proteins [1], Amplification and overexpression of Mcl-1 has been reported in a variety of human tumors (for example, small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer), including hematological malignancies and solid tumors [2,3], Analysis of cancer samples revealed that 36% of breast cancer and 54% of lung cancer samples showed high levels of Mcl-1 expression. Mcl-1 amplification and overexpression are also often associated with poor prognosis and resistance to anticancer drugs. Mcl-1 has been shown to be both an intrinsic and an acquired resistance factor that limits the efficacy of various antitumor agents, including taxol, cisplatin, erlotinib, and other standard anticancer drugs [4], Reducing Mcl-1 expression increases cancer cell susceptibility to drug therapy. Neuroblastoma cell lines with silenced Mcl-1 protein expression have a 2-300- fold increased sensitivity to etoposide, doxorubicin, and ABT-737 [5], Furthermore, silencing of Mcl-1 in in vitro osteosarcoma cell lines and in vivo xenograft tumors has been reported to reverse the chemoresistance of cisplatin and doxorubicin [6], The need for the development of specific Mcl-1 inhibitors has arisen as the number of studies revealing the link between the death resistance of cancer cells and the Mcl-1 protein increases. BAK/BAX, Mcl-1 and other antiapoptotic Bcl-2 proteins, interact with conserved BH3 domains. Therefore, most selective inhibitors are BH3 mimetics. Therefore, the use of peptide (BH3 mimetic peptides) inhibitors developed by mimicking the BH3 domains of proapoptotic proteins has become an effective approach in the inhibition of antiapoptotic proteins. Although BH3 mimetic peptides are highly specific antiapoptotic protein inhibitors, the use of such peptides is limited by their metabolic instability, degradation by cellular proteases, and poor cell permeability [7], BH3 mimetic peptides are not available in the prior art, which, although highly specific, can reach clinical trials.

Many problems are encountered in terms of drug delivery and stability in BH3 mimetic peptides, which play an important role in cancer treatment. Nanoscale drug delivery systems used in the treatment of cancer and many other diseases have become important therapeutic agents as they can be targeted specifically to cancerous tissue and protect the drugs they carry from being destroyed before they reach the target. Nanoparticles hold great promise for overcoming existing barriers in cancer treatment by means of their ability to cross tumor barriers due to their small size and collect in neoplastic tissue. Recent advances have been made with new research on the tumor microenvironment and the design of drug delivery systems that can provide more effective treatments with fewer side effects. However, the application of nanoscale drug delivery systems can successfully overcome the limitations of chemotherapy, reduce the toxicity of drugs, and thus increase the efficacy of antitumor agents.

Peptide drugs, which are frequently used in cancer applications, are considered as new generation drugs due to their high specificity and diversity, especially in targeted therapy [8], In inhibition of Mcl-1 , synthetic peptides have enormous potential as natural inhibitors of protein-protein interactions. Foight et al., in their study, showed that BH3 mimetic structures selectively interact with Mcl-1 and block its antiapoptotic activity in functional analyses. In the same study, they identified the MS1 peptide, the BH3 domain mimetic of the Bim protein that binds very specifically to Mcl-1 and showed that this peptide is at least 40 times more specific than the Bcl-xL, Bcl-2, Bcl-w, and Bfl-1 proteins. Despite such specificity, however, unmodified peptides are poor therapeutic candidates due to their limited sensitivity to in vivo proteolysis and limited capacity to enter cells. Peptides have been chemically modified in order to overcome these disadvantages. For example, BH3 mimetic peptides (SAHB), to which hydrocarbons are added to stabilize it, have been shown to inhibit growth in human leukemia xenografts in vivo [9], However, Barile et al. stated that the solubility of BH3 mimetic peptides they modified by adding hydrocarbon groups decreased and these peptides could not be used in cell experiments. In addition, Okamato et al., in their study, revealed that the target affinity and solubility of the modified peptides decreased.

New drug delivery systems have been developed to promote clinical applications of peptide drugs. In this context, the creation of self-assembled structures has been an effective strategy due to their biocompatibility and ease of synthesis and modification. In recent years, considerable progress has been made in the design of biomaterials with unique self-assembly properties. In most cases, a thermodynamically stable structure is formed through enthalpic and entropic interactions between the basic units of the structure and the reacting solvent molecules. In these structures, electrostatic interactions, hydrophobic interactions, and hydrogen bonds ensure that molecules are stable at low energy levels. The self-assembly process allows the development of many functional materials with desired adjustable properties and structures in one-step design. Peptides can form different nanostructures such as nanotubes, nanofiber and nanovesicles based on their design and self-assembly conditions. Different types and structures of peptides, including dipeptides, amphiphilic peptides, a-helical peptides and [3-layered peptides have been used to form self-assembled nanostructures. In the state of the art, a group of researchers has described the combined use of a cationic domain at the amino-terminus and a histidine-rich region at the carboxy-terminus with a core protein for active utilization of functional peptides. It showed that regardless of the respective origin and amino acid sequence of the core protein, the formed structure resulted in the formation of protein-only circular nanoparticles from 12 to 100 nm. According to this principle, when the amino terminal is labeled with a cationic peptide and the carboxy terminal with a polyhistidine tail, the proteins that form this skeleton cross-react with electrostatic interactions and form toroidal nanostructures that are stabilized by alternating forces such as hydrogen bonding, Van der Waals. These nanoscale materials with editable size enabled the intracellular release of functional proteins to specific target cells and tissues.

It is known in the prior art that many nanomaterials such as liposomes, polymers, dendrimers, and magnetic nanoparticles are also used as drug carriers. Nanopolymers and liposomal systems offer potential in both preclinical and clinical development stages to promote clinical applications of peptide drugs. Nanoparticles effectively encapsulate therapeutic molecules, protecting them from the surrounding pH environment and enzymatic degradation. The small size of the nanoparticles increases the contact area with the epithelial surface, providing great potential for nonspecific uptake or receptor-mediated endocytosis. Antibodies, peptides or small molecular ligands capable of specific interactions with cellular receptors in targeted drug release can be easily conjugated to the surface. By means of all these advantages, polymeric nanoparticles are the carrier systems that are frequently investigated, and their potentials revealed in the targeted release of cancer therapeutics and in combination therapy applications. However, in the use of polymeric nanoparticles, the need for a carrier system such as a polymeric carrier for the delivery of drugs prevents obtaining nanoparticles that are easily prepared in one step and are suitable for scale-up.

In the delivery of peptide-based biomolecules with polymeric, liposomal and dendrimer-based nanoparticle systems, important stability problems may occur during the encapsulation of proteins with these systems. It is known that the organic solvents used can denature proteins. In addition, the difficulties of optimizing nanoparticles that require this carrier, the difficulty of scale-up and the low encapsulation efficiency are among the existing disadvantages.

In the application numbered WO2018049155A1 in the prior art discloses biodegradable polymeric nanoparticles for the delivery of therapeutic agents. Here, biodegradable polymeric nanoparticles are prepared as PLA-PEG-PPG- PEG and used to deliver pharmaceutical agents. It was stated that the nanoparticle obtained here is non-toxic, safe and biodegradable, as well as stable in in vivo studies and has high storage stability. The solution to the present problem here is provided by the encapsulation of the synthetic MS1 peptide into a polymeric nanoparticle carrier system containing the poly(lactic acid)- poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PLA-PEG- PPG-PEG) tetra block copolymer. However, due to the structure of the MS1 peptide in this patent application and the description of a nanoparticle containing a polymeric carrier, disadvantages such as low stability, difficulty in optimization, difficulty in scaling up and low encapsulation efficiency will emerge.

There is a need to develop nanoparticles that are specific to Mcl-1 protein, have high stability and solubility, and do not have difficulties in scaling up and optimizing due to the disadvantages such as low stability, problem in drug portability, difficulty in scale-up and optimization, low encapsulation efficiency, and low resolution in nanoparticle systems used specifically for Mcl-1 protein in cancer treatment in the state of the art.

Brief Description of the Invention

The present invention discloses protein-based nanoparticles, synthesis of these nanoparticles and their use in cancer therapy for improving the stabilization and cellular uptake of MS1 peptide that specifically binds to and inhibits Mcl-1 protein.

In the present invention, a novel fusion protein design has been realized to induce apoptosis in cancer cells. Said fusion protein consists of three parts. The first part is an antiapoptotic inhibitory cationic peptide (MS1 peptide) targeting the BH3 domain of the MCL-1 protein, the second part is a fluorescent protein, and the third part is a peptide containing the amino acid histidine. This engineered protein was expressed and successfully purified by using recombinant DNA technology.

Self-assembled protein-based nanoparticles were obtained from the fusion protein produced with the present invention, and the characterization of the nanoparticles was performed. In an embodiment of the present invention, this protein was also encapsulated into PLGA-PBAE based hybrid polymeric nanoparticles and the characterization of the produced nanoparticles was performed. Nanoparticular systems produced by the present invention showed selective cytotoxic effect against cancer cell line with high MCL-1 expression compared to healthy cell line and induced apoptosis in these cells.

The first object of the present invention is to improve the stability, solubility, and cellular uptake of the MS1 peptide that inhibits Mcl-1 . In this context, MS-1 , which is a selective Mcl-1 inhibitor, is produced recombinantly in fusion with the cationic MS1 peptide, mNeonGreen protein and polyhistidine, resulting in the Bim BH3 mimetic MS1 peptide. In the present invention, self-assembled protein-based nanoparticles are obtained from recombinant MS 1 -mNeonGreen protein. In the present invention, hydrocarbon modifications known in the prior art to cause reduced target affinities and solubility on the peptide are not performed. The peptide becomes more stable and stable than it is individually by means of the production of the peptide of the present invention together with a fusion protein structure and the sequence of this protein and this structure to come together under appropriate conditions to form nanoparticles in a circular structure. Therefore, the present invention does not comprise peptide modifications that are known in the literature for peptide stabilization and have adverse effects; and in addition to the sequences and modifications it comprises, it provides proteinbased nanoparticles with improved stability, solubility, and cellular uptake, by means of the method described in the present invention.

Another object of the present invention is to provide nanoparticles that are easily prepared in one step without the need for any carrier system and are suitable for scale-up. In the present invention, nanoparticle formulations are prepared from the protein purified after bacterial expression, without the need for any carrier system such as a polymeric carrier, only by the dialysis step. Removal of salt and imidazole by dialysis from the protein purified with high salt concentration (500mM) and imidazole (500mM), brings the proteins together. In the present invention, there is no need for a carrier such as a polymer to carry it apart from the protein to be used in therapy. Therefore, the possibility of low encapsulation efficiency in the carrier system and denaturing of the protein during encapsulation processes is eliminated. Thus, by means of the present invention, fast, easy, and stable nanoparticles can be obtained in a single step.

During the preparation of the nanoparticles of the present invention, the nanoparticles of the present invention can be easily prepared and scaled up since there are no parameters such as concentration of carrier polymer, polymer/protein ratio, amount of solvent, mixing speed and time like other nanoparticle systems in the prior art. Conditions such as high temperature and organic solvent are required during the synthesis of carrier materials such as polymers, which exist in the prior art. However, in the present invention, high temperature and the use of organic solvents are not required in the synthesis of the nanoparticle. Stable nanoparticle synthesis is carried out in one step with the ingredients, method steps and dialysis process described in the present invention.

In addition, the dialysis solution disclosed in the present invention consists of sodium bicarbonate and salt. It offers the advantage of being a very environmentally friendly technique compared to the organic solvents used in the preparation of other nanoparticles in the prior art. Particle synthesis is possible in easier and environmentally friendly conditions by means of the present invention since there is no need for devices such as sonicator and homogenizer when creating nanoparticles in the present invention. Another object of the present invention is to obtain nanoparticles that show specific cytotoxic effect to cancer cells and provide Mcl-1 inhibition to be used in cancer treatment. The nanoparticles synthesized by the present invention exhibited selective cytotoxic effect in the HeLa cell line compared to the HLIVEC cell line. Within the scope of the present invention, it has been shown that nanoparticles trigger apoptosis in HeLa cells via the intrinsic pathway. By means of the present invention, new drug delivery systems have been defined that will increase the success of peptide drugs in clinical applications. The resulting nanoparticles reveal nanobiotechnological solutions for the use of peptide-based therapeutic biomolecules. Within the scope of the present invention, it is revealed that the obtained nanoparticles can be used in the apoptosis of cancer cells specifically for Mcl-1 inhibition, thereby providing an effective agent that can be used in the treatment of cancer.

The invention provides nanoparticles that provide Mcl-1 inhibition and improve the stability, solubility and cellular uptake of MS1 peptide, without the difficulties of scale-up and optimization, and cancer treatment agents are provided in which these nanoparticles can be used.

Description of the Figures

Figure 1 : Characterization of MS1-mNeonGreen protein-based nanoparticles (NP). visualization of purified proteins on A. SDS-PAGE with coomassie brilliant blue and analysis with western blot (M. Protein Marker, Co: coomassie brilliant blue staining, WB: western blot), C. Analysis of MS1 NPs and MS1 NPs treated with 1 % SDS by Dynamic light scattering method (DLS), SEM images of D.MS1 NPs at different magnifications.

Figure 2: Characterization of PLGA-PBAE polymeric nanoparticles. A. DLS analysis of PLGA-PBAE NPs and PLGA-PBAE-MS1 NPs, B. SDS-PAGE analysis of MS1 -mNeonGreen protein encapsulated in PLGA-PBAE nanoparticles: M: protein marker, 1 : MS1 - mNeonGreen protein, 2: PLGA-PBAE NPs, 3: PLGA-PBAE-MS1 NPs, C. SEM image of PLGA-PBAE-MS1 NPs.

Figure 3: Graph of the cytotoxicity of MS1 protein and MS1 NPs for HeLa (A) and HUVEC (B) cell lines, cytotoxicity of PLGA-PBAE NPs and PLGA-PBAE- MS1 nanoparticles for HeLa (C) and HUVEC (D) cell lines.

Figure 4: Schematic representation of the MS 1 -mNeonGreen protein. Detailed Description of the Invention

The present invention discloses the formation of self-assembled recombinant protein-based nanostructures containing the MS1 peptide/protein and their use in cancer treatment in order increase cellular uptake and stability of MS1 peptide, which is a selective inhibitor of the antiapoptotic protein Mcl-1 . In order to obtain mentioned protein-based nanostructures, fusion protein containing MS1 peptide was produced recombinantly. In the nanoparticular system which is the subject of the present invention; protein-based nanoparticles are obtained by dialysis from the fusion protein containing the MS1 peptide.

Self-assembled protein nanoparticles, which are the subject of the present invention, are produced from the recombinantly produced Bim protein BH3 domain mimetic MS1 peptide (MS1 -mNeonGreen, recombinant MS1 peptide with the sequence SEQ ID NO:1 ). The schematic representation of the Bim BH3 mimetic MS1 peptide (MS1 -mN eon Green protein) of the present invention is illustrated in Figure 4. Recombinant MS1 peptide with the amino acid sequence of SEQ ID NO:1 comprises MS 1 peptide with amino acid sequence SEQ ID NO:2, anchor peptide with the amino acid sequence of SEQ ID NO: 3, mNeonGreen peptide with the amino acid sequence of SEQ ID NO:4, and polyhistidine peptide with amino acid sequence of SEQ ID NO:5. Here,

• The gene encoding the recombinant MS1 peptide with the amino acid sequence of SEQ ID NO:1 has the nucleotide sequence SEQ ID NO:6,

• The gene encoding the MS1 peptide with the amino acid sequence of SEQ ID NO:2 has the nucleotide sequence of SEQ ID NO:7,

• The gene encoding the linker peptide with the amino acid sequence of SEQ ID NO:3 has the nucleotide sequence of SEQ ID NO:8,

• The gene encoding the mNeonGreen peptide with the amino acid sequence of SEQ ID NO:4 has the nucleotide sequence of SEQ ID NO:9,

• The gene encoding the polyhistidine peptide with the amino acid sequence of SEQ ID NO:5 has the nucleotide sequence of SEQ ID NO: 10, and sequences are shown in the sequence list. Within the scope of the present invention, first of all, the DNA sequence required for the formation of self-assembled peptide nanostructures and the production of the structure shown schematically in Figure 4 was designed. First, the nucleotide sequence was codon optimized according to the E. coli K12 strain; and within framework, the codons in the nucleotide sequence that are low expressed in the microorganism E. coli K12 have been replaced with codons that are more frequently expressed. The jCat codon optimization application was used for codon optimization. Restriction cleavage sites have been added to various regions of the sequence without affecting the codon optimization, and these restriction cleavage sites allow for future modification of the construct and cloning into other expression plasmids as needed. In addition, in the peptide sequence of the present invention, the frequently used restriction cleavage sites were changed without affecting the amino acid sequence and codon optimization.

In an embodiment of the present invention, in the nanoparticular system, the fusion protein poly(D,L-lactide-co-glycolide)-Poly([3-amino ester) (PLGA-PBAE) of the present invention is encapsulated with hybrid nanoparticles. These polymeric nanoparticles, which are an embodiment of the present invention, are obtained with more process steps and in a longer time compared to the proteinbased nanoparticle system, which is the preferred embodiment of the present invention due to the encapsulation process.

The cytotoxicity of MS-1 protein, MS1 protein-based nanoparticles and protein- loaded polymeric nanoparticles on cervical cancer cell line (HeLa) and healthy cell line (Human umbilical vein endothelial cells (HLIVEC)) was analyzed by MTT assay. The apoptotic effect of nanoparticles on HeLa cells was investigated by determining antiapoptotic and proapoptotic protein levels, and as a result, it was revealed that nanoparticles have specific cytotoxic effects against HeLa cells and induce apoptosis in these cells. Process steps, analyzes and obtained results in the synthesis of nanoparticles of the present invention are explained below.

In the recombinant production of MS1-mNeonGreen protein, the synthetic gene encoding the MS1 -mNeonGreen recombinant protein with the nucleotide sequence of SEQ ID NO:6 was designed according to the pET22b vector. Plasmid pET22b-MS1 -mNeonGreen was transferred into E. coli BL21 (DE3) pLysE cells using heat shock. Cells were grown in LB (Luria-Bertani) medium containing 100 pg/mL ampicillin and 34 pg/mL chloramphenicol antibiotic at 37°C and 240 rpm. When the absorbance reached 0.6 at 600 nm wavelength, cells were induced for protein production with 0.1 mM isopropyl-[3-d- thiogalactopyranoside (IPTG).

After induction of protein expression by IPTG, cells were incubated for 3 hours at 37°C at 240 rpm. Cells were then collected by centrifugation (8 000 rpm, 5 min); the resulting cell supernatant was suspended with lysis buffer (20 mM Tris-HCI pH: 8.0, 500 mM NaCI ve 10 mM imidazol), and 100 mM PMSF (Phenylmethylsulfonyl fluoride) and 100 mM Benzamidine were added to the buffer. Cells were lysed with a sonicator for 45 minutes on ice (9 cycles, 100% power, Bandelin Sonopuls HD 2070). The resulting cell lysate was centrifuged at 30 000 rpm for 60 minutes. The MS1 -mNeonGreen protein was purified by IMAC (Immobilized Metal Affinity Chromatography) due to the presence of 6xHisTag in the protein. The lysate was loaded onto the column containing Ni-NTA agarose resin and washed by using 5 column volumes of wash buffer (20 mM Tris-HCI pH: 8.0, 500 mM NaCI ve 20 mM imidazol). Finally, the protein was purified by using elution buffer (20 mM Tris-HCI pH 8.0, 500 mM NaCI and 500 mM imidazole). The purity and integrity of the obtained protein was examined in 12% SDS-PAGE. It was also analyzed by western blotting using the 6xHisTag antibody (1 :5000).

In the present invention, nanoparticle formulations are prepared from the protein purified after bacterial expression, without the need for any carrier system (polymeric carrier), only by the dialysis step. Removal of salt and imidazole by dialysis from the protein purified with high salt concentration (500mM) and imidazole (500mM), brings the proteins together. In the step of preparing selfassembled protein-based nanoparticles, purified MS1 -mNeonGreen protein was filtered through a 0.22 pm pore diameter membrane and dialyzed against 0.16 M NaHCOs and 0.33 M NaCI pH: 7.4 buffer. The dialysis process was carried out in three stages at 250 rpm, and in the first stage, it was kept at room temperature for 1 hour, then at +4 °C for 16 hours and finally at room temperature for 1 hour. After dialysis, the aggregates were removed by centrifugation at 15 000 g for 15 minutes. The obtained MS1 -mNeonGreen self-assembled protein-based nanoparticles (MS1 NPs) were stored at +4 °C.

In an embodiment of the present invention, said protein-based nanoparticles are encapsulated into hybrid polymeric nanoparticles. The polymer used here, poly([3-amino ester) (PBAE), was synthesized and characterized by condensation polymerization via the Michael addition reaction between bisphenol-A-ethoxylate diacrylate and ethylendiamine. PLGA-PBAE hybrid nanoparticles loaded with MS1 -mNeonGreen (PLGA-PBAE-MS1 NPs) were prepared by the oil-in-water (W/O/W) double emulsion solvent evaporation method. 40 mg of PLGA and 10 mg of PBAE were dissolved in 5 mL of DCM (dichloromethane). To the mixture was added 1 mL of MS1 -mNeonGreen (2mg/mL), and a water-in-oil (W/O) emulsion was obtained by mixing with a sonicator at 20% power for 1 minute. This emulsion was added dropwise to 10 mL of 1 .2% PVA solution, and a W/O/W type emulsion was formed by mixing with a sonicator (20% power and 6 cycles, 3 minutes). The emulsion was then diluted with 40 mL of 0.1 % PVA. The resulting nanoparticles were stirred in a magnetic stirrer at 800 rpm for 3 hours at room temperature and the organic solvent was evaporated. Finally, the particles were centrifuged at 10 000 rpm for 15 minutes and dispersed in ultrapure water. The same protocol was followed when preparing blank nanoparticles (PLGA-PBAE NPs). After the nanoparticles were prepared, they were centrifuged at 20,000 rpm for 2 hours at 4 °C. The protein concentration in the supernatant was determined with the BCA protein assay kit. Blank nanoparticles were used as control. The encapsulation efficiency of MS1 -mNeonGreen protein in PLGA-PBAE nanoparticles was calculated by the equation provided below.

Encapsulation efficiency (%)=(Total amount of protein - amount of free protein)/(Total amount of protein) x10

Prepared nanoparticles were evaluated in 12% SDS-PAGE in order to determine the effect of high sonication forces and used organic solvents on the integrity of the MS1 -mNeonGreen protein during nanoparticle preparation. PLGA-PBAE- MS1 NPs, PLGA-PBAE NPs and MS1 -mNeonGreen protein were mixed separately with 2x sample loading buffer. Samples were loaded onto a 12% SDS- PAGE gel after 5 minutes of incubation at 95 °C. The gel was run at 200 V for approximately 45 minutes. Protein bands were stained with Coomassie Brilliant Blue dye and visualized with a gel imaging system.

Then, the mean particle sizes, polydispersity index (PDI) and zeta potentials of the nanoparticles were determined by dynamic light scattering (DLS) at 633 nm (Zetasizer Nano ZS, Malvern Instruments Limited, Malvern). The size and morphology of the nanoparticles were analyzed by Scanning Electron Microscopy (SEM) (TESCAN, MAIA3XMU). SDS-mediated disassembly was performed in order to prove that self-assembled nanoparticles were formed by the assembly of fusion proteins. Self-assembled nanoparticles were incubated for 10 minutes by adding 1 % SDS (sodium dodecyl sulfate) and analyzed by DLS.

During the determination of the physical and serum stability of nanoparticles, the physical stability of the nanoparticles was investigated over a period of 4 weeks. Prepared nanoparticles were kept at +4 °C and particle sizes and zeta potentials were measured at regular intervals for 4 weeks. The serum stability of the nanoparticles was investigated in 10% FBS (fetal bovine serum) for 72 hours. 10% FBS was added to the prepared nanoparticle suspensions and incubated at 37°C. Particle sizes and zeta potentials of the suspensions were measured for 72 hours.

Then, the in vitro cytotoxicity of nanoparticles were investigated on HeLa and HUVEC cell lines. The HeLa cell line was cultured in EMEM medium supplemented with 10% fetal bovine serum, 1 % L-glutamine, 1 % non-essential amino acid, 100 lU/mL penicillin and 10 mg/mL streptomycin. The HUVEC cell line was cultured in DMEM-high glucose medium supplemented with 10% fetal bovine serum, 1 % L-glutamine, 100 lU/mL penicillin and 10 mg/mL streptomycin. All cell lines were cultured at 37°C in a humid atmosphere of 95% air and 5% CO2. In vitro cytotoxicity of nanoparticles were investigated by MTT cell viability assay. Cells were seeded in 96-well plates at a concentration of 5x10 ⁴ cells/mL. After 24 hours, cells were exposed to nanoparticles in a concentration range of 7.8 pg/mL to 500 pg/mL. The cells were then incubated for 72 hours. At the end of this period, the medium was removed from the cells. Then 100 pl of MTT solution was added to each well and incubated for 3 hours at 37 °C in a humid atmosphere containing 95% air and 5% CO2. Formazan crystals produced by cells remaining in MTT solution for 3 hours were dissolved with DMSO and absorbance was measured at 570 nm wavelength. %viability and IC50 values were calculated by using Graphpad Prism 8.01 software.

After examining the cytotoxic effect, changes in expression levels of Bax, Bcl-2, Casp-3, Casp-9, Cyt c, Mcl-1 and p53 genes in HeLa cells treated with synthesized nanoparticles were evaluated by RT-PCR (Bio-Rad CFX96™). Cells were exposed to nanoparticles at IC50 doses for 72 hours. After the incubation period, total RNA was extracted from the cells and cDNAs were synthesized from the RNAs. The relative expression levels of genes were determined using the 2-ΔΔct method.

The Bax, Bcl-2, Casp-3, Casp-9, Cyt c, Mcl-1 , and p53 protein levels in cells exposed to nanoparticles were determined by western blot analysis. HeLa cells exposed to nanoparticles were harvested after 72 hours and RIPA buffer was used for protein isolation. Protein concentration was determined by BCA protein assay. Protein samples (30 pg) separated in 12% SDS-PAGE and were transferred to PVDF membranes using a trans-blot turbo transfer system (BioRad). PVDF membranes were blocked by using 5% skim milk powder (in TBST) for 1 hour at room temperature. The membranes were then incubated with primary antibodies (Anti-Bax (1 :1000), Anti-Bcl-2 (1 :500), Anti-Mcl-1 (1 :1000), Anti-Cyt c (1 :1000). Anti-pro-Casp-3 (1 :500), Anti-pro-Casp-9 (1 :500), Anti-p53 (1 :1000) were incubated at +4°C overnight. After this incubation period, it was washed with TBST and incubated with secondary antibody (goat anti-rabbit IgG H&L (HRP) (1 :10000) for 2 hours at room temperature. After this procedure, protein bands were visualized by using the enhanced chemiluminescence (ECL) substrate, which was washed again with TBST. Anti-GAPDH antibody (1 :5000) was used for normalization and protein expression levels were calculated by using lmageLab 6.1 software.

In the present invention, MS1 -mNeonGreen was successfully produced recombinantly in E. coli and purified in one step by IMAC method. MS1 - mNeonGreen protein was analyzed by coomassie brilliant blue staining and western blot, and its molecular weight was observed to be about 32 kDa as expected (Fig. 1A). In addition, the purified protein is yellow in color and fluoresced under UV light due to the presence of mNeonGreen protein in the structure. The combination of terminal cationic peptides and polyhistidines promotes protein self-assembly. MS1 peptide is highly cationic and the potential of the MS1 -mNeonGreen protein to form self-assembled nanoparticles (MS1 NPs) was investigated. The formation of self-assembled nanoparticles from MS1- mNeonGreen protein was observed in DLS.

The average particle size of MS1 NPs is 20-200 nm, and in an embodiment of the present invention it is in the range of 70-90 nm, preferably 80.57 nm. The polydispersity index (PDI) is in the range of 0-0.5 PDI, and in an embodiment of the present invention it is in the range of 0.3-0.5, preferably 0.394 (Fig. 1 C). PDI indicates the particle size distribution of the nanoparticles, while low PDI values indicate that the particles are monodispersed. PDI values below 0.7 are considered monodisperse. In addition, these nanoparticles were completely disassembled with 1 % SDS and the DLS peak of the monodisperse building blocks was observed (Fig. 1 C). The formation of MS1 -mNeonGreen nanoparticles was also analyzed by SEM. It was observed that the nanoparticles were similar in size to the DLS results and were spherical in shape (Fig. 1 D).

MS1 -mNeonGreen loaded PLGA-PBAE (PLGA-PBAE-MS1 NPs) and empty PLGA-PBAE hybrid nanoparticles (PLGA-PBAE NPs) were prepared by the oil- in-water (W/O/W) double emulsion solvent evaporation method. The sizes of PLGA-PBAE NPs and PLGA-PBAE-MS1 NPs are in the range of 20-200 nm, preferably in the range of 150-170 nm, and PDI values are in the range of 0-0.5. In a preferred embodiment of the present invention, the sizes of PLGA-PBAE NPs and PLGA-PBAE-MS1 NPs were measured as 162.4 nm and 167.2 nm, respectively, according to DLS results. PDI values were measured in the range of 0.076 for PLGA-PBAE NPs and 0-0.5 for PLGA-PBAE-MS1 NPs, and in a preferred embodiment of the present invention, it is measured in the range of 0.05-0.15, preferably 0.094 (Fig. 2A). PDI values indicate that the nanoparticles are highly monodisperse. The efficiency of encapsulation of MS1 -mNeonGreen protein into PLGA-PBAE NPs was calculated as 57±3%.

For an effective formulation, the integrity of the cargo molecule should be maintained. In the present invention, SDS integrity of MS 1 -mNeonGreen protein released from nanoparticles was evaluated by SDS-PAGE in order to determine the effect of high sonication forces and organic solvents used during the preparation of particles. The encapsulation process did not damage the integrity of the MS1-mNeonGreen protein (Figure 2B). When SEM analyzes of PLGA- PBAE-MS1 NPs were examined, it was observed that they were spherical in shape and their dimensions were consistent with the dimensions measured by DLS (Figure 20).

The 4-week stability of the nanoparticle formulations of the present invention was followed. Nanoparticles were prepared and stored at +4±1 °C. Particle size, size distribution and zeta potential of nanoparticles were measured during storage (Table 1 ). No significant changes in particle size or PDI values were observed during 4-week stability studies of nanoparticles. The nanoparticles retained their overall colloidal stability. The zeta potential is a widely used parameter to predict particle stability. A decrease in zeta potential of polymeric nanoparticles was observed during storage. It is predicted that the reduction in the zeta potential of the nanoparticles may be due to the faster degradation of the PBAE polymer than PLGA. Similarly, Sinani et al. reported that the zeta potentials of PBAE polymer with positive surface charge gradually decrease when destabilization begins. In summary, all formulations demonstrated stability at +4±1 °C over a period of 4 weeks.

Table 1. Physical stability of MS1 NPs stored at +4 ± 1 °C.

Serum stability is an important parameter since it determines the strength of the nanoparticles to safely deliver the encapsulated drug to the targeted site and provides an estimate of the in vivo behavior of the nanoparticles. The serum stability of the nanoparticles was monitored for 72 hours by adding 10% FBS to the nanoparticles. Particle size and PDI values were measured by DLS, taking samples at regular intervals. The serum stability of the nanoparticles was quite high. As shown in Table 2, it was determined that the PDI values of the particles increased after 72 hours. Table 2. Serum stability of MS1 NPs.

The cytotoxic activities of the nanoparticles obtained in the present invention were evaluated against HeLa and HLIVEC cell lines using the MTT test. Figure 3 and Table 3 show dose-response curves and calculated ICso values of cell lines after 72 hours of exposure to nanoparticles. Based on the MTT analysis, the synthesized MS1 NPs did not exhibit significant cytotoxic effect against the HLIVEC cell line but did show cytotoxic effect against the HeLa cancer cell line in a dose-dependent manner. In addition, MS1 NPs showed cytotoxic effect at lower ICso values compared to the non-nanoparticle-forming control MS1-mNeonGreen protein (Table 3). The same is true for polymeric nanoparticles. Table 3. IC50 doses of nanoparticles on cell lines.

In the present invention, when RT-PCR analyzes were evaluated to examine changes in antiapoptotic and proapoptotic protein expression levels, a significant increase in Bax/Bcl-2, Casp-9, Casp-3, Cyt c and p53 proteins mRNA levels was observed in HeLa cells exposed to PLGA-PBAE-MS1 NPs and MS1 NPs, while a significant decrease in the mRNA level of Mcl-1 protein was observed. In HeLa cells exposed to the nanoparticles obtained in the present invention, the change in antiapoptotic and proapoptotic protein levels was analyzed by Western blot. In both nanoparticle systems, while there was a significant increase in Bax/Bcl-2, p53 and Cyt c protein levels, there was a decrease in pro-casp-9 and pro-casp-3 levels. RT-PCR and Western blot results substantially supported each other.

Apoptosis is an essential cell death mechanism that plays a vital role in the development and maintenance of cellular homeostasis. A wide variety of intrinsic and extrinsic signals can stimulate intrinsic or extrinsic apoptotic pathways in cells. Members of proapoptotic and antiapoptotic Bcl-2 proteins are directly involved in the control and regulation of intrinsic apoptotic pathways. Release of cytochrome-c from mitochondria is regulated by proapoptotic and antiapoptotic proteins. Then, pro-Casp-9 is activated and cleaved. Activated pro-Casp-9 stimulates Casp-3 and Casp-7 to induce intrinsic apoptotic cell death mechanisms in cells. Upregulation of Bax/Bcl-2 expression ratio and decreased protein level with activation and cleavage of pro-Casp9 and pro-Casp3 are vital biomarkers for the induction of intrinsic apoptosis in cancer cells.

The nanoparticles synthesized with the present invention increased the Bax/Bcl- 2 expression rate and decreased the protein level of Pro-casp-9 and Pro-casp-3. The decrease in pro-casp-9 and pro-casp-3 levels is serious evidence that caspases are activated and turn into active caspase form, initiating apoptosis. According to these results, it has been proven that nanoparticles synthesized within the scope of the present invention activate intrinsic apoptotic cell death mechanisms and exert cytotoxic effects on HeLa cancer cells.

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