Polymer selection for preparation of NPs with high protein loading

Abstract

The present invention relates to novel polymers, which exhibit improved association with protein drugs and enable the formation of nanoparticles (NPs) with high protein loading.

Background

Since the production of bioactive proteins has become feasible in large quantities, there is an increasing need to formulate them into advanced delivery systems that avoid poor patient compliance resulting from the frequent and often invasive administration of these peptide and protein drugs.

One of the most commonly used methods for the delivery of therapeutic proteins, G-CSF included, is the conjugation of the protein with PEG, i.e., the so-called PEGylation. However, PEGylation may induce changes in the physico-chemical properties of the protein, e.g., it may lead to an increase in size and molecular weight of the protein molecule, changes in

conformation, steric hindrance of intermolecular interactions, increased hydrophilicity, and changes in electrostatic binding properties, all of which may affect the pharmacological behaviour of these conjugates. A particular disadvantage is the prolonged circulation time of PEGylated proteins due to a decreased rate of clearance by the kidney, and/or a reduction of proteolysis and opsonisation. Moreover, PEGylation often results in a loss of activity and binding affinity with the intended drug-target, and may further aggregation. All of these disadvantages make the use of PEGylated proteins unattractive and decrease its usability.

Polymeric NPs have been widely investigated as carriers for drug delivery. Much attention has been given to NPs made of synthetic biodegradable polymers such as polycaprolactone, polylactide and polyglycolide due to their biocompatibility. However, these NPs are not ideal carriers for hydrophilic drugs such as peptides and proteins because of their hydrophobic nature, which makes it difficult to prepare a formulation. In particular, the emulsion methods for preparing the NPs typically demand the use of organic solvents, which can irreversibly change the activity of the therapeutic protein. This is why a lot of attention has been recently drawn to NPs prepared from hydrophilic drug carriers, since they do not bind covalently to the protein and NPs can be prepared under milder conditions.

The incorporation of bioactive macromolecules such as proteins into NPs can be achieved by a method based on polyelectrolyte complexation (PEC), which is simple and may be performed under mild conditions. This method involves mixing of the protein and two oppositely charged polymers (polyelectrolytes) in an aqueous medium, which results in spontaneous NP formation. In contrast to the classical high energy (or top-down) methods for producing NPs, the PEC process is driven spontaneously by the intrinsic properties of components and cannot be controlled by formulation parameters. Some known methods also employ ionic crosslinking agents such as tripolyphosphate or calcium chloride to ionically crosslink the polyelectrolytes into NPs. The major driving force of polyelectrolyte complex formation is the electrostatic interaction between oppositely charged polyelectrolyte and protein also known as complex coacervation, and polyionic complexation.

Many combinations of polyanions and polycations were examined for the polyelectrolyte complex formation into hydrophilic NPs. Yet, only a few combinations were found to be suitable for forming stable NPs. Since polyelectrolyte complex formation is purely based on non-covalent, primarily electrostatic interactions, there is a great number of factors that may affect the formation and stability of NPs, e.g., the MW of the polyelectrolytes, stoichiometric ratios and concentration of the polyelectrolytes and the protein, pH, ionic strength, temperature etc. In addition, the capacity of the polyelectrolytes (polymers) for the association with proteins plays a crucial role in the suitability of the NPs as drug delivery systems. The association efficiency ("AE", percentage of protein entrapped in NPs with regard to the initial amount of protein used to produce NPs) and final protein load in the NPs (percentage of protein entrapped in NPs per NP mass) should be sufficiently high to obtain pharmaceutically acceptable systems that can be used further for dosage form formulation, which usually further increases the overall mass of the formulation. Low final protein loading results in excessive amount of formulation with regard to the desired dose for administration.

There is plethora of literature reporting the use of polyelectrolytes for protein delivery, with chitosan (CS) being the most commonly reported polycation in use. The most often reported polyanions associated with CS are natural occurring polysaccharides such as alginate, carboxymethyl cellulose, hyaluronan, heparin, chondroitin sulfate, dextran sulfate etc. and to a lesser extent also polypeptides like polyglutamic acid or polyaspartic acid. For example, US 2008/0254078 and US 2009/01 17195 describe various types of particles that can be prepared from two hydrophilic polymers, namely polysaccharides, one of which exhibits a negative charge (polysaccharide type polyanion or oligoanion), and chitosan or its derivatives exhibiting a positive charge. Anionic polysaccharides may carry carboxymethyl groups (carboxymethyl dextran, carboxymethyl cellulose, carboxymethyl amylose, carboxymethyl beta cyclodextrin), sulfate groups (dextran sulfate and cellulose sulfate) or more than one type of anionic group. For example, glucosamineglucans (GAGs) generally carry carboxy groups and additionally sulfate groups (chondroitin sulfate and heparin). Cationic chitosan derivatives usually used are quaternized derivatives such as A/-tri methyl chitosan, A/-triethyl chitosan or A/-tripropylchitosan. Alternatively, a cationic group can be covalently linked to chitosan, e.g., as in (2-hydroxypropyl trimethyl ammonium) chitosan chloride. US 8354094 describes NPs composed of chitosan and polyglutamic acid for insulin delivery. The NPs prepared had a size of 200-250 nm, a zeta potential of -30 mV and the maximal load of insulin reached with these NPs was 14.2% (AE 55%). These results showed that such type of NPs shelled with CS could transiently open the tight junctions between Caco-2 cell monolayers, and that the insulin loaded NPs could effectively reduce the blood glucose level in a diabetic rat model in vivo.

There are new developments on synthetic, custom made amphiphilic polyelectrolytes. A majority of amphiphilic copolymers has a hydrophilic and a hydrophobic block architecture so that they are able to form nanoscale assemblies (above critical micellar concentration (CMC)) in aqueous media resulting in micelles or vesicles formation. Delivery systems using this type of amphiphilic polymers are usually made by simply mixing the amphiphilic polymer above CMC with protein resulting in micelles formation with relatively low final protein loading. For example, Harada et al. report an amphiphilic copolymer of PEG with glutamic acid modified with n-octanol. Due to the amphiphilic nature of the copolymer, it forms micelles into which the protein was incorporated. Almost 100% of the protein was associated with the micelles, however, 20% of the incorporated protein was simply adsorbed on the surface of micelles and their final loading of G-CSF was only 5% (J Control Rel 201 1 ; 256: 101-108; see also WO 2008/010341 ).

The association of polyelectrolyte complex systems has mostly been studied with insulin or model protein bovine serum albumin (BSA); however their formulation into NPs cannot simply be applied to other proteins as the different proteins may differ considerably in their physico- chemical properties. Therefore, an individual approach for formulating nanoparticulate delivery systems is required for any given bioactive protein.

The incorporation of proteins into nanoscale delivery systems such as nanoparticles (NPs) remains a challenge, and several key requirements need to be observed during the development in order to obtain a suitable nano-sized delivery system. Above all, it is important to preserve the biological activity of the protein, which generally necessitates the use of aqueous media at physiological pH and ionic strength, and at room temperature and applying only mild conditions during the production process, particularly avoiding the use of organic solvents, stabilizers, high shear mixing, homogenisation, elevated temperature etc.; the NPs should preferably have a size of less than 500 nm, and should be colloidally stable; the protein association efficiency (AE) of the NPs should be high in order to obtain a NP formulation with a high final protein load per NP mass.

There is hence an ongoing need for an effective delivery system for bioactive proteins. The present invention provides new polymers, which allow for the preparation of NPs having high association efficiency (AE) and a high protein loading capacity, and which can be readily formulated further into oral dosage forms with improved pharmacokinetics of the proteins. Figure Legends

Figure 1: Overlap of H NMR spectra of sodium poly(glutamic acid), containing different amounts of octylamine: lowest line - P(Glu-oa) 4%, middle line - P(Glu-oa) 13% and upper line - P(Glu- oa) 24%.

Figure 2. Average scattering intensity of complexes formed between polyglutamate-octylamine polymers (P(Glu-oa)) and G-CSF prepared with two different initial concentrations of P(Glu-oa), 2 mg/ml (A) and 3 mg/ml (B).

Figure 3. Association efficiency of G-CSF in complexes with P(Glu-oa) polymers with 4%, 13% and 24% octylamine grafting at initial polymer concentration of 2 mg/ml and 3 mg/ml and with different initial G-CSF loading of 100 μΙ and 200 μΙ (4.4 mg/ml).

Figure 4. Particle size (A) and average scattering intensity (B) of NPs prepared by step-wise addition of TMC to P(Glu-oa)/G-CSF complexes. Initial concentration of P(Glu-oa) was 2 mg/ml.

Figure 5. Particle size (A) and average scattering intensity (B) of NPs prepared by step-wise addition of TMC to P(Glu-oa)/G-CSF complexes. Initial concentration of P(Glu-oa) was 3 mg/ml.

Figure 6. Association efficiency of G-CSF in NPs prepared with P(Glu-oa) polymers with 4%, 13% and 24% octylamine grafting at initial polymer concentration of 2 mg/ml and 3 mg/ml and with different amount of TMC added. The G-CSF was kept constant (100 μΙ, 4.4 mg/ml).

Detailed description

For the development of suitable NPs for protein delivery, it is important to preserve the biological activity of protein (which is assured by mild and aqueous conditions of NPs production), and to prepare small (preferably below 500 nm) and colloidally stable NPs with sufficiently high protein association with polymers that results in NP formulation with high final protein load per NPs mass. High association efficiency (AE) does not necessarily results in high final loading (FL) (see comparative example 1 ) which is, however, crucial in the development of suitable NPs for protein delivery. In order to obtain suitable NPs for protein delivery having these properties, careful selection of the polymers is important.

The present invention provides novel polymers that exploit multiple types of interactions (electrostatic, hydrophobic interactions and hydrogen bonds) with protein drugs. It has been surprisingly found that the polymers of the present invention exhibit improved association efficiency (AE) for protein drugs and allow for the assembly of NPs with high final protein loading (FL). The polymers provided herein do not form large associates with proteins, which results in lower probability of complex precipitation.

The polymers of the invention possess a suitable molecular weight and charge, and are either low molecular weight polymers having an anionically charged backbone and hydrophobic grafts, or are high molecular weight polymers having polysaccharide backbone and anionic grafts. Suitable low molecular weight polymers according to the invention are, for example, polyamino acids with hydrophobic grafts; suitable high molecular weight polymers according to the invention are, for example chitosan polymers with hydrophilic polyanionic grafts.

The molecular weight of the low molecular weight polyamino acid based polymers of the invention can be up to about 12 kDa, or up to about 1 1 kDa, or up to about 10 kDa, or up to about 9 kDa, or up to about 8 kDa, or up to about 7 kDa, or up to about 6 kDa, or up to about 5 kDa, or up to about 4 kDa, preferably, the molecular weight is about 4 kDa to about 12 kDa, or from about 4 kDa to about 10 kDa, or from about 4 kDa to about 9 kDa, or from 4 kDa to about 8 kDa, or from 4 kDa to about 7 kDa, or from 4 kDa to about 6 kDa, or from 4 kDa to about 5 kDa. The molecular weight of the higher molecular weight polymers based on chitosan grafted with hydrophilic polyanionic chains of the invention should be above about 60 kDa, or above about 70 kDa, or above about 80 kDa, or above about 90 kDa, preferably, the molecular weight is about 60 kDa to about 130 kDa, or from about 70 kDa to about 120 kDa, or from about 80 kDa to about 1 10 kDa, or from 90 kDa to about 100 kDa.

The polyamino acid polymers of the invention are grafted with hydrophobic moieties such as alkylamine chains having from 2 to 20 carbon atoms, or from 6 to 16 carbon atoms, or from 8 to 12 carbon atoms or any other hydrophobic amines. The chains may be the same or different, it is however preferred that the polyamino acids are grafted with one kind of hydrophobic chains. Preferred hydrophobic chains are octylamines.

The degree of polyamino acid grafting with alkylamines plays an important role in the complexation (nanoparticle formation) process, as the hydrophobic chains on the polyamino acid polymer contribute significantly to protein association (Fig. 1 A). The association efficiency (AE) for NPs increases with increasing grafting of polyaminoacids (e.g., polyglutamate) with alkyl chains (e.g., octylamines). Polyamino acids such as polyglutamates are suitably grafted with more than about 4% alkylamine chains, such as octylamine, or more than about 5%, 10%, 13%, 16%, 19%, 22%, 25%, 28%, 30%, 35%, 40%, 45%, 50% or more. Suitably, polyglutamate is grafted with about 4% to about 50%, or about 4% to about 35%, or about 4% to about 30% or about 4% to about 24%, or about 13% to about 24% octylamine. While a high degree of grafting of polyglutamate with octylamine is generally beneficial, the degree of grafting should not exceed a value where the octylamine grafted polyglutamate is no longer water soluble. The final properties of protein association and NP formation can be fine-tuned by varying the degree of grafting.

The higher molecular weight polymers based on chitosan are grafted with hydrophilic chains such as polyamino acids, e.g., with polyglutamates. Suitably, for the higher molecular weight polymer, the chitosan : polyglutamate (Chi:PGIu) ratio is from 1 :1 to 1 :10, e.g., 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8 or 1 :9. The final properties of protein association and NP formation can be fine- tuned by varying the degree of grafting. Preferred polymers of the invention are selected from polyglutamate grafted with octylamine (P(Glu-oa)), particularly polyglutamate having a molecular weight of about 5 kDa to about 8 kDa grafted with about 4% to about 30% octylamine; higher molecular weight polymers based on chitosan grafted with polyglutamate (Chi-g-PGIu), particularly higher molecular weight polymers based on chitosan grafted with polyglutamate having a molecular weight of above about 60 kDa and having a (Chi:PGIu) ratio from 1 :1 to 1 :10; and mixtures thereof. The polymers of the invention allow for the formation of NPs having a particularly high final protein loading per NP mass. A second oppositely charged polymer may complement the NP formation. A suitable polymer in this regard is trimethylchitosan (TMC).

Due to the unique characteristics of these polymers, they are able to hierarchically assemble in certain concentration ranges (below the concentration where self-association of said polymer(s) occurs) with bioactive proteins into nano-sized systems with greatly improved protein association efficiency in comparison to other polyelectrolytes such as the ones presented as comparative examples of the current state of the art in present application, resulting in a high final protein load (at least 10%) of the resulting NPs, and minimal loss of protein during production.

Conventional high MW polyelectrolytes (natural-like) usually entrap the protein either physically or through electrostatic interactions, which typically results in poor protein association. Without being bound to any theory, it is believed that the improved protein association attained with the polymers provided herein is achieved through the exploitation of specific macromolecular interactions between polymer and protein such as electrostatic interactions, hydrophobic interactions and hydrogen bonding, which is believed to be a result of defined polymer chemistry and architecture of these polymers.

The new polymers provided herein are water soluble and therefore enable formation of the NPs in aqueous media without using organic solvents, which are damaging to protein drugs. In addition, the present polymers enable the formation of NPs spontaneously after stepwise addition of the protein solution to the polymer solution under gentle stirring, which makes this method particularly suited for sensitive protein drugs.

Nanoparticles

The present invention further provides nanoparticles comprising one or more polymers according to the present invention together with one or more proteins. The examples provided herein show that when the protein is associated with one or more of the present polymers, this results in the formation of nanoparticles with very high protein association efficiency and high final protein load, which is enhanced over the protein load of NPs of the prior art.

In one embodiment, the invention provides nanoparticles comprising one or more proteins, and one or more polymers selected from low molecular weight polyamino acids grafted with alkyl chains as defined above, low molecular weight polyglutamate grafted with octylamines as defined above, and higher molecular weight polymer based on chitosan grafted with

polyglutamates as defined above. In one embodiment, the invention provides nanoparticles comprising one or more proteins, and a low molecular weight sodium polyglutamate grafted with hydrophobic octylamine as defined above and a higher molecular weight polymer based on chitosan grafted with polyglutamate as defined above. While these two polymers may be present at any ratio, it is preferred that both polymers are present in equal amounts at a ratio of about 1 : 1. An oppositely charged polymer such as trimethylchitosan TMC may further be present in said nanoparticles. Other suitable oppositely charged polymers are known to one skilled in the art.

The average particle size of the NPs provided herein is below 600 nm for optimized formulation parameters, preferably from 200 to 600 nm, from 200 to 500 nm, from 200 to 400 nm, or from 200 to 300 nm.

The final protein load of the NPs provided herein is at least 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30%.

It has been surprisingly found that besides the electrostatic interactions, the hydrophobic interactions of the polymers of the present invention significantly contribute to complex formation. Such types of protein-loaded NPs are preferred over NPs formed by electrostatic interaction only (prepared usually from polysaccharides: polyanions and polycations) or based on micelles (prepared from amphiphilic polymers)).

The terms peptide and protein are used herein interchangeably. Any protein is principally suited to be associated with one or more polymers of the present invention; a particularly suited protein is G-CSF. The term "G-CSF" as used herein refers to Granulocyte-Colony Stimulating Factor and may be a natural or recombinant G-CSF, recombinant human G-CSF (rhG-CSF), or any protein or peptide having in vivo biological activity of the G-CSF glycoprotein, e.g., a wild type or mutant G-CSF, a G-CSF peptidomimetic, or a G-CSF fragment. The species from which G-CSF is derived can be animal, mammal or human species. Human G-CSF is, however, preferred. Human G-CSF comprises the known human Granulocyte-Colony Stimulating Factor having 174 amino acids. It further comprises human G-CSF in its naturally glycosylated state, i.e., including all carbohydrate side chains, in particular glycosylation at Thr133. The abbreviation "G-CSF" as used herein stands for G-CSF as defined above. Other proteins equally suited to be associated with one or more polymers of the invention are proteins selected from erythropoietin, interferon alpha, interferon beta, HGH (human growth hormone), and other cytokines.

Method of preparing nanoparticles

General Method of polvelectrolvte complexation (PEC) for nanoparticle formation The procedure of NP preparation is important to ensure formation of defined NP size, stable dispersion, efficient protein entrapment in NPs (association efficiency, AE) and high protein load per NP mass.

Polyelectrolyte complexes (nanoparticles) are generally formed by a two-step process. Briefly, a protein solution is added in a drop-wise manner to a solution comprising one or more polymers according to the present invention to give a mixture, in which a complex formation between the protein and the one or more polymers takes place. During this step, the pH of the protein solution is typically below the isoelectric point of the protein, so that the protein is positively charged; the concentration of the polymer solution comprising one or more polymers of the invention should not exceed the limit value at which self-association of polymer(s) occurs. The complex thus formed may be in the form of nanoparticles, and the resulting mixture may be a dispersion of nanoparticles. After stirring for a defined time, during which said complex is formed between the protein and the one or more polymers of the invention, a second polymer may be added, which has a charge that is opposite the net charge of said complex. The second polymer may improve the process of protein entrapment in NPs. The use of trimethylchitosan (TMC) as the second, positively charged polymer bears the advantage of being able to disturb self association of the polymer(s) of the invention, so that higher concentrations of said polymer(s) can be employed.

An efficient NP formation takes place when a low molecular weight P(Glu-oa) polymer with a higher content of octylamine side chains is used (such as defined above). If a high molecular weight chitosan polymer provided herein is used, the ratio (Chi:PGIu) should be from 1 :1 to 1 :10. The presented results show the important contribution of hydrophobic interaction to effective association of the polymers with protein. The addition of trimethylchitosan (TMC) as highly positively charged polyelectrolyte to the polymers of the invention, e.g., to P(Glu-oa)/protein, or Chi-g-PGIu/protein, or Chi-g-PGIu/P(Glu-oa)/protein further improves NP formation.

Characterisation of nanoparticles

The particle size and polydispersity index of NPs were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS ZEN 3600 (4mW He-Ne laser, 633 nm) from Malvern instruments, UK. Scattering light was detected at 173° by the automatically adjusted laser attenuation filters and the measurement position within the cell at 25°C. For data analysis, the viscosity (0.8863 mPa) and refractive index (1.330 at 633 nm) of the distilled water at 25°C were required. Particle diameter measured by DLS is a value that reflects the diffusion of spherical particle within the fluid and, thus, represents a hydrodynamic diameter of a spherical particle.

Polydispersity index (Pdl) is a parameter defining particle size distribution of NPs. It is a dimensionless number extrapolated from the autocorrelation function and ranges from values close to zero for the uniform particles' size distribution and up to values close to 1 for the broad size distribution of particles. Average scattering intensity or average count rate, which is the measure of NP's concentration in the sample, was also monitored during the experiments. This parameter represents the scattering intensity of the sample in the absence of laser attenuation filters (adjusters of the laser power). It can be used for the comparison in scattering intensity (kcps) between the samples and indicates the concentration of particles in the sample. The average count rate is a calculated value obtained from the measured count rate divided by the attenuation factor, as shown in the following expression: Derived count rate = (measured count rate)/(attenuation factor).

Zeta potential of particles was also measured with the same instrument using a technique of electrophoretic light scattering (ELS) also called laser Doppler velocimetry, in combination with phase analysis light scattering. Scattering light was detected at an angle of 13° by the automatically adjusted laser attenuation filters and the measurement position within the cell at 25°C. For data analysis a viscosity, refractive index and dielectric constant (ε = 78.5 at 25°C) of water at 25°C were used.

Using the Zetasizer Nano software, the quality report message was also checked after each measurement. It is displayed as a warning message with notes "result meets or does not meet quality criteria". This report reflects the suitability of the sample for DLS/ELS measurement, and the reliability of the data obtained, since it is based on a collection of measurement parameters that need to be examined to obtain an adequate degree of confidence regarding the

measurement result.

Association efficiency and final protein loading in complexes or NPs

Association efficiency of granulocyte colony-stimulating factor (G-CSF) in complexes/NPs was determined indirectly after separating complexes/NPs with associated G-CSF from the dispersion media containing non-associated G-CSF. The prepared dispersions (examples 1 , 2, 3, 4, 5, 6) were centrifuged at 40.000 rpm for 20 min and the supernatant was analysed for the content of free protein with RP-HPLC.

Efficiency of G-CSF association (AE - association efficiency) with polymers was calculated by the difference between the total amount of G-CSF used to prepare the complexes/NPs and the amount of G-CSF present in aqueous phase after centrifugation.

, ,_,„. total amount of GCSF - unassociated GCSF

AE % = x 100

total amount of GCSF The amount of G-CSF associated with complexes/NPs was also calculated and is referred to as the final protein loading (FL). It represents the difference between the total amount G-CSF initially used to prepare the complex/NPs and the amount of non-associated G-CSF as a percentage of the total amount of complexes/NPs.

total amount of GCSF - unassociated GCSF

total amount of complexes

Examples of the current state of the art

Comparative Example 1

NPs made of alginate, G-CSF and trimethylchitosan (TMC)

The NPs for the delivery of G-CSF protein were prepared using two of the most common oppositely charged polyelectrolytes, alginate and chitosan derivate, i.e., trimethylchitosan (TMC). They were prepared in medium with pH 4.5 - below the isoelectric point of protein G-CSF.

Alginate was used as the first negatively charged polyelectrolyte to which G-CSF was added drop-wise, and then the second positively charged polyelectrolyte TMC was added in the same way to form NPs.

Table 1 shows the results of different experiments of NPs formation in which the amounts of G- CSF and TMC added were varied. When high amount of G-CSF was added to the alginate solution, precipitation of the particles occurred, which results in an inappropriate-unstable system. When the amount of added G-CSF was decreased (25 μΙ, 4.4 mg/ml) complexes of alginate/G-CSF in the size of 700 nm were formed. In the following experiments, TMC was added stepwise to alginate/G-CSF complexes, which progressively resulted in smaller, more defined particles. In addition, the average scattering intensity of sample increased by the addition of TMC, which reflects efficient NPs formation.

The association efficiency and final protein loading in NPs was also determined for some of the samples with different TMC additions (Table 1 ). The association efficiency of G-CSF in NPs increased for the formulations prepared with more TMC polymer and ranged from 44% to 71 %. These percentages of protein entrapment in NPs were quite satisfactory, however considering the amount of all polymers used to prepare NPs the final protein loading in NPs was calculated to be very low. Final G-CSF loading in NPs was achieved only up to 2%. Table 1: Results of NPs formation of G-CSF with alginate and TMC.

Pdl: Polydispersity index; AE: Association efficiency; FL: Final G-CSF loading in NPs

Comparative Example 2

NPs made of lower molecular weight polymer based on chitosan-graft-polyglutamate (Chi-g-PGIu LMW), G-CSF and TMC

Scheme 1 : Synthetic pathway for preparation of Chi-g-PGIu LMW.

Chitosan (Chi) (M _n = 18.9 kDa, M _w = 34.2 kDa, D _M = 1 -8, degree of deacetylation = 99 %, viscosity declared by the manufacturer 7 mPa.s) was transformed by (±)camphor-10-sulfonic acid (CSA) into chitosan (±)camphor-10-sulfonate (Chi CSA).

Chi CSA (1 eq.) was reacted with γ-benzyl-L-glutamate A/-carboxyanhydride (BGIu NCA) (30 eq.) in dry DMSO under dry inert atmosphere. Chitosan-graff-poly(L-benzyl glutamate) (Chi-g- PBGIu ₃₀ ) was collected as a fine white powder.

Chi-g-PBGIu ₃₀ (1 eq.) was dissolved in trifluoroacetic acid (TFA) at room temperature and then HBr/acetic acid (33 % w/w) (4 eq.) was added. After 2 hours the reaction mixture was poured into excess of cold diethyl ether. The resulting suspension was centrifuged, washed and dried. The solid was then dissolved in water by adjusting pH to 8-9 by adding 0.1 M NaOH. The clear solution was dialyzed and freeze-dried to obtain Chi-g-PGIu LMW (M _n = 7.23 kDa, M _w = 8.92 kDa, D _M = 1 -2, glutamate:glucosamine ratio = 30:1 ). Preparation of NPs of G-CSF with Chi-q-PGIu LMW and TMC

Chi-g-PGIu LMW was dissolved in water (in concentrations of 3 mg/ml and 5 mg/ml), and the solution pH was adjusted to pH 5.7 in order to prepare NPs below the isoelectric point of G-CSF (pi 6.1 ). The Zeta potential of polymer solution after adjusting pH was -25 mV. The solution of G-CSF (4.4 mg/ml in buffer pH 4.5) was added drop-wise to Chi-g-PGIu LMW solution (1 ml, 3 mg/ml or 5 mg/ml). During addition of G-CSF the solution became cloudy as a consequence of complex formation. The size of complexes was in the range of app. 170 - 300 nm, depending on the polymer concentration and amount of G-CSF added (Table 2). Then, the second positively charged polymer, i.e., trimethylchitosan (TMC) was added, which results in formation of larger particles. Average scattering intensity increased progressively by the addition of TMC. In all cases well defined particles were formed.

The association efficiency and final protein loading in NPs were determined for different formulations. Results showed that efficiency of protein association for this type of NPs was very low ranging from 0 to 22%, and the final G-CSF load in NPs accounts to a maximum of 1.5% for the optimal formulation.

Table 2: Results of NP formation of G-CSF with Chi-g-PGIu LMW and TMC.

Pdl: Polydispersity index; AE: Association efficiency; FL: Final G-CSF loading in complexes Examples of the present invention

We prepared a new set of anionic polymers based on sodium polyglutamate which was modified with different amounts of octylamine chains (P(Glu-oa)). These polymers are able to form different types of interactions with the protein drugs (electrostatic, hydrophobic interactions, hydrogen bonds). In addition, the P(Glu-oa) have a rather low average molecular weight (M _p = 5 kDa as determined by MALDI-TOF) and consequently they do not form large associates with proteins, which results in lower probability of complex precipitation.

Three sodium polyglutamates with different degrees of hydrophobic modifications were prepared:

· polyglutamate-octylamine 4% (4% of polyglutamate -COOH are modified with alkyl chains) - P(Glu-oa) 4%,

• polyglutamate-octylamine 13% (13 % of polyglutamate -COOH are modified with alkyl chains) - P(Glu-oa) 13%,

• polyglutamate-octylamine 24% (24 % of polyglutamate -COOH are modified with alkyl chains) - P(Glu-oa) 24%.

Synthesis of alkyl modified sodium polyglutamates

Aminolvsis of polv(y-benzyl L-qlutamate) (P(BLG))

Molar mass characteristics of P(BLG) used were: M _n = 7.6 kDa, M _w = 8.0 kDa and M _w /M _n = 1.05. P(Glu-oa) 4%:

P(BLG) (1.00 g, 4.57 mmol) and 2-hydroxypyridine (2-HP) (0.43 g, 4.57 mmol) were dissolved in dry A/,A/-dimethylformamide (DMF, 15.5 mL). Octylamine (375 μΙ_, 2.26 mmol) was then added and the reaction mixture was stirred for 48 hours at room temperature. Afterwards, the reaction mixture was poured into cold distilled water to precipitate the product. The precipitate was collected by filtration, washed with water and dried in vacuum. Final product (P(BLG)-oa)) was washed with hexane to remove the remaining octylamine (0.797 g, Yield = 73%).

P(Glu-oa) 13%:

A analogous procedure was used as for P(Glu-oa) 4% above using P(BLG) (1.00 g, 4.56 mmol), 2-HP (0.43 g, 4,56 mmol), octylamine (1515 μΙ_, 9.1 mmol), DMF (15.2 mL), 24 hours, (0.934 g, 85%).

P(Glu-oa) 24%:

A analogous procedure was used as for P(Glu-oa) 4% above using P(BLG) (0.50 g, 2.28 mmol), 2-HP (0.22 g, 0.28 mmol), octylamine (1890 μΙ_, 1 1.4 mmol), DMF (15.5 mL), 24 hours, (0.318 g, 58%). Deprotection of P(BLG-oa)

P(Glu-oa) 4%:

P(BLG-oa) (0.81 g, 3.71 mmol) was dissolved in TFA (20 mL). Then, HBr/acetic acid (33% w/w) was added (3.5 mL). The reaction mixture was stirred for 1 hour. Subsequently, it was poured into Et ₂ 0 to precipitate the product, which was collected by filtration. The product was washed with water to remove remaining HBr and was finally freeze-dried (0.395 g, 83%).

P(Glu-oa) 13%:

A analogous procedure was used as for P(Glu-oa) 4% above using P(BLG-oa) (0.85 g, 3.87 mmol), TFA (21.2 mL), HBr/acetic acid (3.6 mL), (0.846 g, 74%).

P(Glu-oa) 24%:

A analogous procedure was used as for P(Glu-oa) 4% above using P(BLG-oa) (0.33 g, 1.52 mmol), TFA (8.4 mL), HBr/acetic acid (1 .4 mL), (0.177 g, 91 %).

All products were finally neutralized with 0.1 M NaOH to convert them into water-soluble alkyl modified sodium polyglutamate (P(Glu-oa)) and then dialysed to remove excess NaOH.

Scheme 1: Synthesis of octyl modified sodium polyglutamates.

Water-soluble alkyl modified sodium polyglutamates were prepared with different degrees of alkylation (Table 3) as calculated from H NMR (Figure 1 ). The degree of alkylation was calculated from the integrals of the signal for methyl group of alkyl chain (signal 1 ) and methyne groups in polymer backbone (signal 3).

Characteristics of P(Glu-oa) polymers in water at various pH values are presented in table 3. Table 3. Characteristics of P(Glu-oa) polymers in water, buffer pH 4.5 and buffer pH 7.

These polymers were titrated with either G-CSF only or G-CSF followed by trimethylchitosan and the complexes formed were characterized by dynamic/static light scattering technique using Zetasizer from Malvern instruments.

Example 3

Complexes of octylamine modified polyglutamate and G-CSF

The octylamine modified polyglutamate polymers, having overall negative charge, were first titrated with G-CSF solution in a medium with pH 4.5, i.e., below the isoelectric point of G-CSF protein (pi 6.1 ). At this pH the protein G-CSF is positively charged. The results are presented in Figure 2.

Stepwise addition of G-CSF solution (4.4 mg/ml) to solution of P(Glu-oa) polymers (2 mg/ml and 3 mg/ml) resulted in gradual increase in the scattering intensity, indicating complex formation between G-CSF protein and P(Glu-oa) polymers. However, the size of complexes formed could not be properly determined, most probably due to the formation of complexes of different sizes.

The degree of polyglutamate grafting with octylamine plays an important role in complexation process. In the case of 2 mg/ml P(Glu-oa) concentration, the scattering intensity of formed complexes increases with increasing degree of grafting (P(Glu-oa) 24% > P(Glu-oa) 13% » P(Glu-oa) 4%). These results reveal that hydrophobic alkyl chains on the polyglutamate polymer contribute to protein association significantly (Fig. 1 A). For P(Glu-oa) 4%, containing low amount of alkyl chains, only a small increase in scattering intensity was observed, indicating that beside electrostatic interaction also the hydrophobic interaction is significant for complexation formation.

The efficiency of complexation between G-CSF and P(Glu-oa) 13% or P(Glu-oa) 24%

(measured as scattering intensity) diminished when P(Glu-oa) was used in concentrations higher than 3 mg/ml (Fig. 1 B), indicating the importance of the initial P(Glu-oa) concentration for successful complexation. At higher polymer concentration, both P(Glu-oa) most probably associates itself, which reduces the probability of P(Glu-oa) to interact with the protein. The addition of higher amounts of G-CSF (200 μΙ) to 1 mL P(Glu-oa) 13% or P(Glu-oa) 24% solution of 3 mg/ml concentration did not improve complexation efficiency between the polymer and the protein. On the contrary, the P(Glu-oa) 4% polymer showed similar NPs formation independent of initial polymer concentration (2 mg/ml or 3mg/ml), which reveals the role of alkyl chains in the self-association of P(Glu-oa)13% and P(Glu-oa) 24%.

Association efficiency and final G-CSF load in complexes

Efficiency of protein association in complexes is presented in Figure 3 and Table 4, for different polymer concentrations (2 mg/ml and 3 mg/ml) and for different G-CSF additions (different initial G-CSF loadings). The percentages of AE varied from 30 to 90% depending mostly on the degree of alkylation and polymer concentration, whereas the amount of G-CSF (initial G-CSF loading) minimally influenced association efficiency.

The lowest %AE were obtained for polymer P(Glu-oa) 4% (30-40%), which was quite expected from the results obtained by DLS measurements (Figure 2), indicating poor complex formation in the case of P(Glu-oa) 4% polymer. For other two polymers (P(Glu-oa) 13% and P(Glu-oa) 24%) with higher ocytlamine grafting, the association efficiency reached very high values of approximately 80 and 90%. These results clearly indicated that octylamine grafts on the polymer chain significantly contributes to protein association. In addition, protein association was always lower for the P(Glu-oa) polymer concentration of 3 mg/ml which is in line with DLS results of complex formation, indicating poorer complexation of G-CSF with P(Glu-oa) polymers at high polymer concentration (Figure 2).

The G-CSF loading in complexes is presented in Table 4, together with association efficiency. The final loading of G-CSF in complexes increased with degree of octylamine grafting and is higher for complexes prepared with higher amount of G-CSF added. A comparison of %AE as well as %FL for the complexes prepared with different degree of alkyl chain grafting on polyglutamate reveals much higher difference between the P(Glu-oa) 4% and the P(Glu-oa) 13 % than between the P(Glu-oa) 13% and the P(Glu-oa) 24%. As expected, lower values of %AE and %FL were obtained for the complexes prepared at 3 mg/ml than at 2 mg/ml polymer concentration.

G-CSF loading in complexes was the highest for P(Glu-oa) polymers with 2 mg/ml concentration and the highest initial protein load tested (30.6%) and reached the values of 1 1 %, 26% and 27% for polymers P(Glu-oa) 4%, P(Glu-oa) 13% and P(Glu-oa) 24%, respectively. Table 4. Association efficiency (AE) and final G-CSF loading (FL) in complexes.

Pdl: Polydispersity index; AE: Association efficiency; FL: Final G-CSF loading in complexes

Example 4

NPs made from octylamine modified polyglutamate, G-CSF and TMC

For preparation of NPs from P(Glu-oa)/G-CSF complexes, the TMC solution was added to P(Glu-oa)/G-CSF solution in a final step. For these experiments we used P(Glu-oa) polymers in a concentration of 2 mg/ml or 3 mg/ml with 100 μΙ G-CSF (4.4 mg/ml). After complex formation the TMC (3 mg/ml) was added in increment of 10 μΙ. The results presented in Figures 4 and 5 indicate particle size and average scattering intensity of particles prepared with P(Glu-oa) polymer in concentration of 2 mg/ml (Fig. 4) or 3 mg/ml (Fig. 5) and with increasing addition of TMC. Results are presented only for those samples that yielded stable nanodispersion.

By the addition of TMC to P(Glu-oa)/G-CSF the scattering intensity increased progressively for polymers P(Glu-oa) 13% and 24% whereas much lower scattering, which did not changed with increasing amount of TMC added, was detected for the P(Glu-oa) 4% polymer (Fig. 4B). The extent of NP formation was thus more pronounced for the polymers containing higher amount of octylamine grafts, indicating the importance of hydrophobic groups in the polymer. The highest value was achieved with P(Glu-oa) 24%, having the highest octylamine grafting. Interestingly, at higher 3 mg/ml polymer concentration the efficiency of NP formation progressively increases with increasing amount of TMC added up to the same extent as for the 2 mg/ml P(Glu-oa) polymer concentration (Fig 5B). This indicates that higher P(Glu-oa) concentration did not hinder particle formation which was the case for complex formation of P(Glu-oa) with G-CSF. As expected, higher amount of TMC had to be added to 3 mg/ml than to 2 mg/ml P(Glu-oa) polymer before the precipitation of particles occurred. These results reveal that TMC is able to disturb self-associated P(Glu-oa) macromolecules at higher polymer concentration and, as a consequence can form stable NPs through electrostatic interaction.

For P(Glu-oa) 13% and P(Glu-oa) 24% polymers the particle size was in the range of 200 to 600 nm and did not change much by progressive TMC addition, which reflects efficient packaging of polymers. On the contrary, the P(Glu-oa) 4% showed different results. The particle size increased by the addition of TMC up to 1200 nm, whereas the scattering intensity remained quite the same throughout the titration experiment and never exceeded 20,000 kcps. Therefore, polymers P(Glu-oa) 13% and 24%. showed even higher potential for formulation preparation as compared to the P(Glu-oa) 4% polymer.

Association efficiency and final G-CSF load in NPs

The addition of TMC to P(Glu-oa)/G-CSF complexes results in a well-defined nanodispersion. The efficiency of protein association in NPs for different polymer concentration (2 mg/ml and 3 mg/ml) and different amount of TMC addition, which resulted in stable nanodispersion is presented in Figure 6 and Table 5. In all experiments the G-CSF added to P(Glu-oa) solutions was kept constant (100 μΙ, 4.4 mg/ml).

The bars in Figure 6 show that association efficiency (AE) for NPs increases with increasing grafting of polyglutamate with alkyl chains. Again better association was achieved for 2 than for 3 mg/ml P(Glu-oa) concentration. The AE values reached up to 85% and 93% for P(Glu-oa) 13% and P(Glu-oa) 24% polymer, respectively. The P(Glu-oa) 4% polymer showed poorer association efficiency, which ranges from 37 to 54%. These results indicate improved G-CSF association efficiency when TMC was used for NP formation, whereas the difference in AE between formulations containing different amount of TMC is negligible.

For the prepared P(Glu-oa)/G-CSF/TMC NPs the final G-CSF loading was also calculated (Table 5). The FL depends on the initial amount of protein and polymer used for NPs preparation. The highest FL values were obtained for the P(Glu-oa) polymers containing high amounts of octylamine side chains (13 and 24 %) and for the polymer concentration of 2 mg/ml.

Table 5. Association efficiency (AE) and final G-CSF loading (FL) in P(Glu-oa)/G-CSF/TMC NPs. For comparison purposes, the AE and FL of G-CSF in P(Glu-oa)/G-CSF complexes are added.

P(Glu-oa) 4% 100 0 31 4.3 (3 mg/ml) 10 12.7 282 0.243 14.924 54 7.3

30 12.5 696 0.404 34.062 42 5.6

60 12.2 1 107 0.427 17.557 37 4.9

P(Glu-oa) 100 0 75 13.8 13%

10 17.8 325 0.486 73.497 70 13.2

(2 mg/ml)

30 17.4 366 0.456 105.990 85 15.2

P(Glu-oa) 100 0 52 7.1 13%

10 12.7 332 0.648 22.661 69 9.1

(3 mg/ml)

30 12.5 404 0.392 49.477 69 8.9

60 12.2 409 0.400 67.860 73 9.2

P(Glu-oa) 100 0 89 16.4 24%

10 17.8 570 0.574 1 10.283 93 16.8

(2 mg/ml)

30 17.4 474 0.523 147.170 93 16.4

P(Glu-oa) 100 0 65 8.7 24%

10 12.7 284 0.510 41.090 78 10.2

(3 mg/ml)

30 12.5 422 0.471 96.230 76 9.8

60 12.2 292 0.299 1 14.334 77 9.6

Pdl: Polydispersity index; AE: Association efficiency; FL: Final G-CSF loading in complexes

Example 5

Synthesis of higher molecular weight polymer chitosan-graft-polyglutamate Chi-g-PGIu

Chi-g-PBGIu-i5 was prepared under similar procedure from the same batch of the starting chitosan material as described in comparative Example 2. Chi CSA (1 eq.), BGIu NCA (15 eq.), DMSO (79 mL). Yield: 94 %, M _n = 4.79x 10 ⁵ Da, M _w = 9.61 * 10 ⁵ Da, D _M = 2.0.

For deprotection of carboxyl groups of PBGIu grafts the Chi-g-PBGIu ₁₅ (475 mg, 2.0 mmol, 1 eq.) was dissolved in DMF (20 mL) and chilled to 0 °C. 40 % w/w solution of tetrabutylammonium hydroxide (TBAH) in water (2 mL, 1.5 eq) was added and the reaction mixture was stirred. After reaction completion the TBAH was quenched and the product was precipitated by addition of 1 M AcOH. The suspension was centrifuged and the precipitate was dissolved in minimal amount of saturated aqueous NaHC0 ₃ followed by dialysis. The resulting solution was freeze-dried to obtain Chi-g-PGIu HMW as a white fluffy solid. Yield: 54 mg (16 %), M _n = 5.76x 10 ⁴ Da, M _w = 9.04x 10 ⁴ Da.

NPs made of higher molecular weight polymer chitosan-graft-polyglutamate (Chi-g-PGIu HMW), G-CSF and TMC

Polymers Chi-g-PGIu HMW with different chitosan:polyglutamate ratio (Chi:PGIu=1.1 and 1 .8) was dissolved in water in concentration 2 mg/ml and all solutions of polymer have pH below the isoelectric point of G-CSF (pi 6.1 ). The Zeta potential of polymer solution were -24 and -37 mV for Chi:PGIu ratio 1.1 and 1 .8, respectively.

NPs were prepared as follows: the polymer solution (1 ml, 2 mg/ml) was titrated with the solution of G-CSF (4.4 mg/ml in buffer pH 4.5) in increment of 100 μΙ. During addition of G-CSF all solutions became cloudy. The size of complexes was in range of app. 200 - 250 nm, depending on added G-CSF quantity and the Chi:PGIu ratio. Then the positively charged trimethylchitosan (TMC) was added drop-wise. The addition of TMC was found to have negligible influence on the particle size. In all cases well defined particles were formed.

The association efficiency (AE) of protein in complexes and NPs is presented in Table 6, for different Chi:PGLu ratios. The addition of G-CSF was the same for all experiments. The association efficiency varied from 67% to 82%, depending of Chi:PGIu ratio and TMC addition. The AE% values were similar with or without TMC addition, when Chi-g-PGIu HMW polymer with Chi:PGIu ratio 1.1 was used but TMC had an influence on AE% when Chi-g-PGIu HMW polymer with Chi:PGIu ratio 1.8 was used. For the prepared Chi-g-PGIu HMW/G-CSF complexes and Chi-g-PGIu HMW/G-CSF/TMC NPs the final G-CSF loading was also calculated (Table 6). The final loading was very high regardless of Chi:PGIu ratio or TMC addition and was approximately 36% - 42%.

Table 6. Association efficiency (AE) and final G-CSF loading (FL) in Chi-g-PGIu HMW/G- CSF/TMC NPs. For comparison purposes, the AE and FL of G-CSF in Chi-g-PGIu HMW/G-CSF complexes are added.

*Chi:PGIu ratio; Pdl: Polydispersity index; AE: Association efficiency; FL: Final G-CSF loading in complexes

Example 6:

NPs made of a mixture of octylamine-modified-polyglutamate(P(Glu-oa), higher molecular weight polymer chitosan-graft-polyglutamate(Chi-g-PGIu HMW) (P(GI-oa)-Chi-g-PGIu HMW), G-CSF and TMC

Two polymers were combined, octylamine-modified-polyglutamate (P(Glu-oa)) and higher molecular weight polymer chitosan-graft-polyglutamate (Chi-g-PGIu HMW). The use of such polymer solution allowed protein to interact electrostatically and hydrophobically with P(Glu-oa) and/or electrostatically as well through polar interactions with the polysaccharide backbone of Chi-g-PGIu HMW. NPs were obtained by dropwise addition of G-CSF (4.2 mg/ml in buffer pH 4.5) into the solution of P(Glu-oa) and Chi-g-PGIu HMW (total polymer concentration was 2 mg/ml in water, where P(Glu-oa) -Chi-g-PGIu HMW ratio was 1 :1 and 1 :2 and pH for both solutions were 5.6). After gentle stirring, trimethylchitosan (3 mg/ml in water) was slowly added to form NPs of uniform particle size between 200 nm and 250 nm and low polydispersity.

The P(Glu-oa)-Chi-g-PGIu HMW ratio plays an important role in complexation process. When concentrations of both polymers were equal (1 : 1 ) the average scattering intensity was higher comparable to those where more Chi-g-PGIu HMW was used (P(Glu-oa)-Chi-g-PGIu HMW ratio was 1 :2). The increasing amount of TMC added to P(Glu-oa)-Chi-g-PGIu HMW/G-CSF complexes also increased the scattering intensity and particle size in samples with both P(Glu- oa)-Chi-g-PGIu HMW ratios.

The protein association efficiency in NPs with different P(Glu-oa)-Chi-g-PGIu HMW ratio and different amount of TMC addition is presented in Table 7. The values in Table 7 show that AE for NPs increases with the amount of TMC addition. The AE values were higher for NPs prepared with mixture of polymers (P(Glu-oa) and Chi-g-PGIu HMW) in comparison to AE where only Chi- g-PGIu HMW polymer was used to prepared NPs (Table 6). The AE values were higher when equal amount of P(Glu-oa)-Chi-g-PGIu HMW and more TMC were used and reached up to 98.7 %.

For the prepared P(Glu-oa)-Chi-g-PGIu HMW/G-CSF/TMC NPs the final G-CSF loading was also calculated (Table 7). The final G-CSF loading was found to be between 24% and 39%. Table 7. Association efficiency (AE) and final G-CSF loading (FL) in P(Glu-oa)-Chi-g-PGIu HMW/G-CSF/TMC NPs.

Pdl: Polydispersity index; AE: Association efficiency; FL: Final G-CSF loading in complexes

Conclusions of the experiments

From the titration experiments presented in this invention we concluded that more efficient NP formation took place when P(Glu-oa) polymer with higher content of octylamine side chains was used. However, the initial polymer concentration should not exceed the limit value at which the self-association of P(Glu-oa) polymer occurred (> 3 mg/ml). The presented results showed the important contribution of hydrophobic interaction to effective association of P(Glu-oa) polymers with G-CSF protein. The addition of trimethylchitosan as highly positively charged polyelectrolyte to P(Glu-oa) G-CSF further improves NP formation, indicating the fact that electrostatic interaction contributed to complexation process as well.

For the titration experiments with anionic polymer Chi-g-PGIu HMW and positively charged protein G-CSF we obtained excellent AE and FL as well, showing that we can achieve good NP formation based on electrostatic interaction between Chi-g-PGIu HMW polymer and G-CSF and together with the polysaccharide backbone of Chi-g-PGIu HMW polymer and G-CSF. The reason that addition of TMC does not increase the AE and FL, as it is the case with P(Glu-oa), might be, that the interaction between the Chi-g-PGIu HMW polymer and TMC is stronger than the interaction between polymers and protein thus excluding protein from the nanoparticles. The presented results also showed that the mixture of both anionic polymers together with the TMC allow us even better association of polymers with protein and higher protein association in NPs.