POLYMER-CARGO-COMPLEXES COMPRISING CROSS-LINKED COPOLYMERS AND CARGO MOLECULES

Abstract

Polymer-cargo-complexes including cross-linked copolymers and cargo molecules bound to the copolymers by electrostatic interactions. The non-medical use thereof for transfection and to a kit including such polymer-cargo-complexes. The polymer-cargo-complexes for use in therapy, in particular for use in gene therapy or peptide/protein drug delivery. A method of preparing the polymer-cargo-complexes. A kit for preparing a polymer-cargo-complex of the invention.

Claims

1. A polymer-cargo-complex comprising: a) a cross-linked copolymer, the copolymer comprising two alternating units A and B forming a repeat unit A-B such that the copolymer comprises a (A-B).sub.n backbone with n being the number of repeat units of the backbone, wherein unit A is a derivative of an amino acid hydrazide and unit B is a derivate of a dialdehyde comprising a polyethylene glycol (PEG) group, wherein 40 to 100 mol % of unit A are derivatives of hydrazides of either cationic amino acids selected from the group consisting of lysine (Lys), arginine (Arg), histidine (His) and combinations of two or more thereof, or anionic amino acids selected from the group consisting of aspartic acid (Asp), glutamic acid (Glu) and combinations thereof, wherein the copolymer comprises imine groups and acyihydrazone groups altematingly linking together the alternating units A and B of the polymer backbone such that the units A and B of the backbone are each linked to one neighboring unit by an imine group and to the other neighboring unit by an acyihydrazone group, and b) cargo molecules bound to the cross-linked copolymer by electrostatic interactions between the cargo molecules and the amino acid side chains of unit A.

2. The polymer-cargo-complex according to claim 1, wherein the copolymer comprises cross-linking groups linking together distinct units A such that one cross-linking group links together two units A.

3. The polymer-cargo-complex according to claim 2, wherein the ratio of the total number of cross-linking groups to the total number of units A of the polymer is in a range of from 0.05:1 to 0.45:1.

4. The polymer-cargo-complex according to claim 1, wherein the PEG group is a PEG chain having from 3 to 12 units.

5. The polymer-cargo-complex according to claim 1, wherein the cargo molecule is selected from the group consisting of nucleic acids and peptides.

6. The polymer-cargo-complex according to claim 1, wherein the dialdehyde comprising a PEG group is a carbazole dicarboxaldehyde comprising a PEG group.

7. The polymer-cargo-complex according to claim 1, wherein 0 to 60 mol % of unit A are derivatives of hydrazides of amino acids selected from the group consisting of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gin), cysteine (Cys), glycine (Gly), proline (Pro), alanine (Ala), valine (Val), isoleucine (lie), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and combinations of two or more thereof.

8. The polymer-cargo-complex according to claim 1, wherein the polymer-cargo-complex is a nanoparticle having a hydrodynamic diameter (DH) in a range of from 50 nm to 350 nm.

9. A non-therapeutic use of a polymer-cargo-complex of at least claim 1 for transfection.

10. The polymer-cargo-complex of claim 1 for use in therapy.

11. The polymer-cargo-complex according to claim 10 for use in gene therapy or peptide drug delivery.

12. The polymer-cargo-complex according to claim 10 for use in treatment and/or prevention of viral diseases.

13. A kit comprising a polymer-cargo-complex, or amino acid hydrazides and dialdehydes comprising a PEG group for preparing a polymer-cargo-complex of claim 1.

14. A method of preparing a polymer-cargo-complex according to claim 1 comprising the following steps: a) Providing an aqueous solution comprising (i) hydrazides of amino acids and (ii) dialdehydes comprising a PEG group, wherein the solution has a pH in the range of from 2 to 6, and wherein 40 to 100 mol % of the hydrazides are hydrazides of either cationic amino acids selected from the group consisting of lysine, arginine, histidine and combinations of two or more thereof, or anionic amino acids selected from the group consisting of aspartic acid, glutamic acid and combinations thereof, b) Incubating the solution to allow copolymer formation, c) Mixing the formed copolymer with a cargo molecule in an aqueous solution, d) Optionally adding amino acid side chain-specific cross-linker to the solution, e) Increase the pH of the solution to at least 7.

15. The method according to claim 14, wherein the cargo molecules of step c) are nucleotides, and wherein the nucleotides are added such that the NP ratio is from 1 to 50.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0116] FIG. 1 is a bar graph showing the hydrodynamic diameter OH of a copolymer (Lys-biodynamer) obtained from Lys-hydrazide and PEGGylated carbazole dicarboxaldehyde. The y-axis shows the average OH (±standard deviation) determined by DLS. The copolymer concentration is shown on the x-axis. The results are shown for two different pH conditions.

[0117] FIG. 2 is a bar graph showing cell viability in presence of different concentrations of copolymers. In addition to Lys-biodynamers, biodynamers obtained from Hishydrazide and PEG6ylated carbazole dicarboxaldehyde (His-biodynamers) and from Arg-hydrazide and PEG6ylated carbazole dicarboxaldehyde (Arg-biodynamers) have been tested as well Prior art compound PEI was used as a control. The copolymers of the invention have strongly reduced cytotoxicity as compared to the prior art. Cytotoxicity was tested in A549 cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

[0118] FIG. 3 is a bar graph showing the hydrodynamic diameter OH of a polymer- cargo-complex nanoparticle formed by electrostatic interaction of the Lys-biodynamer with mCherry mRNA. The y-axis shows the average (±standard deviation) deter- mined by DLS. The nanoparticle concentration is shown on the x-axis. The results are shown for two different pH conditions (pH 5.0 (AcOH) and pH 7.4 (HBSS), respectively).

[0119] FIG. 4 shows transfection efficiency (of mCherry mRNA) and toxicity of PEI and polymer-cargo-complexes of the present invention. A549 cells were incubated for 2 hours with different transfection reagents (present invention and prior art). Subsequent- ly, the cells were washed and further incubated for 24 hours in a cell culture medium for protein expression, then followed by flow cytometry measurement for evaluation of transfection efficiency and cell viability. Transfection efficiency was assessed by mCher- ry fluorescence. Cell viability was assessed using an amine reactive dye (DAPI (4′, 6-diamidine-2′-phenylindole dihydrochloride)) resulting in weakly stained non-permeable live cells and more highly fluorescent dead cells due to increased permeability of the membranes. Buffer and mRNA without transfection agents were used as negative controls. Significant transfection was calculated by multiplying the percentage of transfected cells with the percentage of viable cells. The significant transfection using polymer-cargo-complexes of the present invention is about 20 times higher as compared to prior art PEI.

[0120] FIG. 5 shows DLS data showing the size distribution of polymer-cargo-complexes of the invention. The x-axis depicts the hydrodynamic diameter D.sub.H of the polymer-cargo-complexes (in nm) on a logarithmic scale. The y-axis depicts the intensi- ty (in %). FIG. 5A shows the results of polymer-cargo-complexes having mCherry mRNA as model nucleic acid cargo molecule. The Z-average hydrodynamic diameter was determined to be 120 nm. The polydispersity index (PDI) was 0.174. FIG. 5B shows the results of polymer-cargo-complexes having albumin-fluorescein isothiocyanate conjugate as model peptide cargo molecule. The Z-average hydrodynamic diameter was determined to be 244 nm. The polydispersity index (PDI) was 0.333.

[0121] FIG. 6 shows transfection efficiency (of mCherry mRNA) and toxicity of poly-mer-cargo-complexes of the present invention. As polymer-cargo-complexes, mCherry encoding mRNA complexed with KHR-BDy was used. DC2.4 cells were incubated for 2 hours with the polymer-cargo-complexes in various cell culture medium conditions as indicated in FIG. 6. Subsequently, the cells were washed and further incubated for 24 hours in the indicated cell culture medium conditions for protein expression, then followed by flow cytometry measurement for evaluation of transfection efficiency and cell viability. Transfection efficiency was assessed by mCherry fluorescence. Cell viability was assessed using an amine reactive dye (DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride)) resulting in weakly stained non-permeable live cells and more highly fluorescent dead cells due to increased permeability of the membranes. Untreated cells were used as control. Significant transfection was calculated by multiplying the percentage of transfected cells with the percentage of viable cells. The y-axis shows the results for the different conditions (±standard deviation).

[0122] FIG. 7 shows DLS data showing the size distribution of polymer-cargo- complexes of the invention. The x-axis depicts the hydrodynamic diameter DH of the polymer-cargo-complexes (in nm) on a logarithmic scale. The y-axis depicts the scattering intensity (in %). FIG. 7 shows the results of polymer-cargo-complexes (DH=126 nm, PDI=0.321) of positively charged Arg-Phe-biodynamer (RF-biodynamer) with negatively charged insulin as cargo molecule.

[0123] FIG. 8 summarizes properties of polymer-cargo-complexes formed from Lysbiodynamer and a model siRNA, alexa-594-conjugated siRNA. FIG. 8A is a bar graph showing the hydrodynamic diameter DH of a polymer-cargo-complex nanoparticle formed by electrostatic interaction of the Lys-biodynamer with alexa-594-conjugated siRNA. The y-axis shows the average (±standard deviation) determined by DLS. The nanoparticle concentration is shown on the x-axis. FIG. 8B is a bar graph showing the encapsulation efficiency (in % (w/w)) for two different NP ratios (±standard deviation).

[0124] FIG. 9 shows cellular uptake of the polymer-cargo-complexes formed from Lysbiodynamer and Alexa594-labelled siRNA. A549 cells were incubated for 2 hours with different transfection reagents (present invention and prior art). Cellular uptake was assessed by fluorescence-activated cell sorting. HBSS buffer and free siRNA without co-polymer of the invention were used as negative controls. The y-axis shows the cellular uptake (±standard deviation).

Examples

[0125] Preparation of the copolymer

[0126] General methods and instrumentation

[0127] All reagents were obtained from commercial suppliers without further purification. Procedures were not optimized regarding yield. NMR spectra were recorded on a Bruker AV 500 (500 MHz) spectrometer. Liquid chromatography-Mass spectrometry was performed on a SpectraSystems-MSQ LCMS system (Thermo Fisher, Dreieich, Germany). Flash chromatography was performed using the automated flash chromatography system CombiFlash Rf+(Teledyne Isco, Lincoln, NE, USA) equipped with RediSepRf silica columns (Axel Semrau, Sprockhovel Germany) or Chromabond Flash C18 columns (Macherey-Nagel, Duren, Germany). The purity of compounds synthe-sized by us was determined by LC-MS using the area percentage method on the UV trace recorded at a wavelength of 254 nm and found to be >95%.

[0128] Synthesis

[0129] The following scheme is an overall synthesis scheme of a particular preferred copolymer.

##STR00008##

[0130] In the following, the different synthesis steps are described in more detail.

[0131] Synthesis of compound b1

##STR00009##

[0132] Carbazole dialdehyde (b1) was prepared according to a literature procedure (J. F. Folmer-Andersen, E. Buhler, S.-J. Candau, S. Joulie, M. Schmutz, J.-M. Lehn, Polym. Int. 2010, 59, 1477 and D. A. Patrick, D. W. Boykin, W. D. Wilson, F. A. Tanious, J. Spychala, B. C. Bender, J. E. Hall,C. C. Dykstra, K. A. Ohemeng, R. R. Tidwell, Eur. J, Med. Chem, 1997, 32, 781-793), Briefly, 3,6-dibromocarbazole (3.02 g, 9.29 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 60 ml) to give a pale brown solution. The solution was set stirring in a dry ice/acetone bath. An amount of 26 mL of a solution of n-butyllithium (n-BuLi) (2.5 mM in hexane, 65.03 mmol) was then added over a period of 10-15 minutes, causing the reaction contents to become light yellow. The cooling bath was removed for 1 h and then replaced back. After 10 min, anhydrous dimethylfor- mamide (DMF, 7.55 ml, 97.54 mmol) was added over 10 min, causing the precipitation of a pale yellow solid. The cooling bath was removed and the reaction was stirred for 90 more minutes. After this, it was quenched with 1M hydrochloric acid (HCl) solution and the reaction was suction filtered. The filtrate was extracted with ethylacetate (EtOAc, 5×50 ml) and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate (Na.sub.2SO.sub.4). The orange oily product was purified via cornbiflash column chromatography (dichloromethane (DCM) : methanol 97:3) yielding 260 mg (15%) off-white solid. 1H-NMR is in agreement with those previously reported (J. F. Folmer-Andersen, E. Buhler, S.-J. Candau, S. Joulie, M. Schmutz, J.-M. Lehn, Polym. Int. 2010, 59, 1477 and D. A. Patrick, D. W. Boykin, ‘A’. D. Wilson, F. A. Tanious, J. Spychala, B. C. Bender, J. E. Hall, C. C. Dykstra, K. A. Ohemeng, R. R. Tidwell, Eur. J. Med. Chem. 1997, 32, 781-793).

[0133] Synthesis of compound B1

##STR00010##

[0134] Compound 1)1 (1.27 g, 5,69 mmol) is dissolved in 70 ml of DMF. Tosylated hexaethylene glycol monometyl ether (compound b2, m is 6 in this example) prepared according to a literature procedure (J. F. Folmer-Andersen, E. Buhler, S,-J. Candau, S. Jodie, M, Schmutz, J.-M. Lehn, Polym. Int, 2010, 59, 1477 and a A. Patrick, D. W. Boykin, W. D. Wilson, F, A. Tanious, J. Spychala, B. C, Bender, J. E. Hall,C, C. Dykstra, K. A. Ohemeng, R. R, Tidwell, Eur. J. Med. Chem. 1997, 32, 781-793) Compound b2 (2.50 mg, 5.55 mmol) was added to this solution followed by potassium carbonate (K2003, 2.54g, 18,38 mmol). 10 mg of sodium iodide (Nal) is added to this mixture and the reaction was stirred at 80 ° C. to reflux overnight. Extraction was done with DCM and the organic phase was washed with brine. The compound was purified using flash chromatography with ethylacetate/propanol (5:1) 2.22 g product was obtained as a pale yellow liquid which occasionally solidified upon standing, (Yield 80%) The 1H-NMR of the product agreed with previous reports (J. F. Folmer-Andersen, E. Buhler, S.-J. Candau, S. Joulie, M. Schmutz, J.-M. Lehn, Polym. Int. 2010, 59, 1477 and D. A. Patrick, D. W. Boykin, W. D. Wilson, F. A. Tanious, J. Spychala, B. C. Bender, J. E. Hall,C. C. Dykstra, K. A. Ohemeng, R. R. Tidwell, Eur. J. Med. Chem. 1997, 32, 781-793).

[0135] Synthesis of compound A1

##STR00011##

[0136] Monomer A (R.sub.1 is a side chain of lysine in this example, A1) is prepared according to a literature procedure (Y. Liu, M. C. A. Stuart, E. Buhler, J.-M. Lehn, A. K. H. Hirsch, Adv. Punct. Mater. 2016, 26, 6297). To a solution of the Lysine methyl ester hydrochloride (750 mg, 3.2 mmol) in methanol (15 mL), hydrazine monohydride (25.8 mmol) was added. The reaction mixture was stirred at 25 00 for 20 h. The mixture was then concentrated and dried overnight in a high vacuum. After lyophilization, L-lysine hydrazide was obtained as liquid (495 mg, 96 %) .sup.1H-NMR is in agreement with those previously reported (Y. Liu, M. C. A. Stuart, E. Buhler, J.-M. Lehn, A. K. H. Hirsch, Adv. Fund. Mater. 2016, 26, 6297).

[0137] Synthesis of the copolymer (biodynamer) from monomer A1 and B1

[0138] Each monomer was dissolved in 100 mM d-acetate buffer (pa 5.0) in a final concentration of 20 mM, The monomer solutions were mixed with a ratio of 50:50. After 24 hours of reaction at room temperature (r.t.), the resulting mixture was filtered using a 0.22 μm polyethersulfone (PES) syringe filter. Polymerization was confirmed by consumption of the aldehyde proton peaks, analyzed by 1H-NMR, and nanorod formation was observed by dynamic light scattering (DLS). 1H-NMR peaks of the biodynamer agreed with the previous report.

[0139] Formulation scheme of the polymer-cargo-complex

[0140] The resulting copolymers (biodynamers) in an acidic aqueous solution diluted to 100 pg/mL with deionized water. Nucleic acids or proteins were added to the biodynamer solution with a calculated amount based on the NP ratio or the zeta potential, respectively, and vortexed for 3 seconds. In particular, the copolymer (zeta potential=16.7 mV) was mixed with cargo having negative zeta potential (e.g. luciferase siRNA (2 mg/mL) =−7.99 +−3.8 mV in 50 mM acetate buffer pH 5 or albumin (2 mg/mL albuminfluorescein isothiocyanate conjugate, Merck, Germany) =−10.04+−0.82 mV in 50 mM acetate buffer pH 5), Regarding luciferase siRNA as cargo molecule, the amount as used was such that the NP ratio was 10:1. Regarding the peptide cargo molecule (albumin-fluorescein isothiocyanate conjugate), the amount as used was 5 wt.-% as compared to the amount of copolymer (biodynamer). Polymer-cargo-complexes were formed as nanoparticles. The formed nanoparticles (polymer-cargo-complexes) stabilized at r.t. for 1 hour before characterizations or uses.

[0141] Crosslinking scheme

##STR00012##

[0142] After the polymer-cargo-complex formulation, a crosslinking agent (e.g., glutaraldehyde) crosslinked amino acid side chains to improve particle stability, Glutaraldehyde solution (16%) was diluted 100 folds with deionized water. The diluted glutaraldehyde solutions were added into the nanoparticle solutions with pL scales. The exact amount of the glutaraldehyde was calculated based on the w % of the biodynamers. The resulting mixture was vortexed for a few seconds and stored at r.t. overnight.

[0143] Hydrodynamic size of nanoparticles

[0144] Copolymers were produced by the method of the invention using Lys-hydrazide (A1) and PEG6ylated carbazole dicarboxaldehyde (B1) as described above.

[0145] It was found that the copolymers formed nanoparticles in solution. The hydrody-namic diameter (D.sub.H) of the nanoparticles was determined using DLS. As shown in FIG. 1, at a pH of 7.4 (10 mM phosphate buffer) the DH of the nanoparticles was independent of the concentration of the copolymers in the solution. In contrast, at a pH of 5,0 (in 10 mM acetate buffer) the Z-average hydrodynamic diameter DH of the nanoparticles decreased with decreasing concentration of MDs. Thus, there is degradation of the copolymer at acidic conditions whereas the copolymer is stable at neutral pH values.

[0146] Formation of polymer-cargo-complexes comprising the copolymer and nucleic acid or peptide cargo molecules

[0147] The OH substantially increased upon addition of nucleic acids (mCherry mRNA) or peptides (albumin-fluorescein isothiocyanate conjugate), indicating formation of polymer-cargo-complexes comprising the copolymer and nucleic add or peptide cargo molecules, respectively.

[0148] Polymer-cargo-complexes of the invention having nucleic adds as cargo mole- cules may also be termed polyplexes. Polyplex formation was done in deionized water. The DH was determined to be about 120 nm by DLS with a polydispersity index (PDI) of 0.174 (FIG. 5A).

[0149] Notably, the DH was substantially constant between about 100 nm and about 200 nm at different copolymer concentrations at pH 7.4. In contrast, the DH substantially increased with decreasing nanoparticie concentration at pH 5.0 up to almost 900 nm at a concentration of 3.1 μg/ml. This indicates that controlled release of the cargo is possible at low pH. The results are shown in FIG. 3.

[0150] Regarding polymer-cargo-complexes having albumin-fluorescein isothiocyanate conjugate as peptide cargo molecule, the DH was determined to be about 244 nm by DLS with a polydispersity index (PDI) of 0.333 (FIG. 5B).

[0151] To further confirm the potential of polymer-cargo-complexes as a therapeutic protein carrier, complex formation was tested using insulin as cargo molecule. Insulin is a peptide hormone available as a therapeutic protein for diabetes. Positively charged ArgPhe-biodynamer (RF-biodynamer) formed polymer-cargo-complexes with negatively charged insulin. FIG. 7 is a dynamic light scattering result showing hydrodynamic size and size distribution of the respective polymer-cargo-complexes.

[0152] Low cytotoxicity

[0153] Cytotoxicity was tested in A549 cells using the MTT assay. Copolymers based on Lys, His or Arg derivatives were compared with prior art reagents PEI.

[0154] Different concentrations of the reagents were incubated with A549 cells (10,000 cells per well in 96 well plates) for 24 hours. The results are shown in FIG. 2 as average±standard deviation. The copolymers of the invention have strongly reduced cytotoxicity as compared to the prior art.

[0155] High transfection efficiency

[0156] A549 cells (100,000 cells per well) were incubated for 2 hours with different transfection reagents (present invention and prior art) comprising nucleic acids corre- sponding to 0.5 pg mRNA per well. The mRNA used was mCherry mRNA. Subsequent- ly, the cells were washed and further incubated for 24 hours in a cell culture medium (10 FCS (fetal calf serum) containing RPM (Roswell Park Memorial Institute) 1640 medium) for protein expression, then followed by flow cytometry measurement for evaluation of transfection efficiency and cell viability. Transfection efficiency was assessed by mCherry fluorescence. Cell viability was assessed using an amine reactive dye (DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride)) resulting in weakly stained non- permeable live cells and more highly fluorescent dead cells due to increased permeability of the membranes.

[0157] Buffer and mRNA without transfection agents were used as negative controls. PEI (polyethylene imine, cationic polymeric transfection agent) was used as prior art control. Copolymers of the present invention were tested as follows: [0158] 200 pg/mL copolymers, 20 NP ratio based on Lys derivatives (20 Lys-BDy), [0159] 200 pg/mL copolymers, 20 NP ratio based on mixture of Lys, His and Arg derivatives (20 KHR-BDy),

[0160] Regarding KHR-BDy, the molar fractions of Lys, His and Arg were, 40%, 30% and 30%, respectively.

[0161] The results are shown in FIG. 4. Significant transfection was calculated by multiplying the percentage of transfected cells with the percentage of viable cells. For example, if 50% of viable cells are transfected and viability is 80%, the significant transfec- tion is 0.5−0.8=0.4=40%. Thus, the significant transfection indicates the percentage of transfected cells based on the total cell number (dead+alive). Cytotoxicity is an important parameter when evaluating the transfection efficiency, not only because of further in vivo application but also because the toxicity affects actual transfection efficiency. The present invention is particularly advantageous based on the low toxicity and high significant transfection efficiency.

[0162] In fact, the significant transfection is about 20 times higher as compared to PEI (the gold standard of polymeric transfection agents) considering 20 Lys-BDy and 20 KHR-BDy. Notably, particularly high significant transfection efficiency was achieved with 20 KHR-BDy, thus with copolymer comprising derivatives of Lys, His and Arg.

[0163] Transfection of dendritic cells

[0164] To test the potential of polymer-cargo-complexes as a vaccine, the transfection ability and toxicity was tested using dendritic cells (DC2.4). The results are shown in FIG. 6.

[0165] Dendritic cells are one of the major antigen-presenting cells, processing antigen material and present it on the cell surface to the T cells of the immune system.

[0166] As a polymer-cargo-complex, mCherry encoding mRNA was complexed with KHR-BDy.

[0167] The polymer-cargo-complexes transfected dendritic cells with a transfection efficiency of 100%.

[0168] The toxicity was varied by the transfection condition. When the transfection condition was closer to the physiological condition, cell viability increased. The DC2.4 cell viability was 20% in an isotonic buffer (HESS) but increased up to 79% in cell culture media (RPMI-1640) containing 10% of serum protein (fetal calf serum, FCS). Thus, the polymer-cargo-complexes of the present invention are particularly effective under physiological conditions indicating their suitability as vaccines.

[0169] Formation of polymer-cargo-complexes of the copolymer and siRNA cargo molecules

[0170] Complex formation of the copolymer was tested using siRNA as cargo molecule. siRNA has a potential for cancer treatment, and gene therapy by regulating protein expressions of target cells.

[0171] Lys-biodynamer formed polymer-cargo-complexes having a hydrodynamic diameter of 150 nm to 230 nm with a model siRNA, alexa-594-conjugated siRNA. The results are shown in FIG. 8A.

[0172] Encapsulation efficiency was dependent on the NP ratio as shown in FIG. 8B. At 20 NP ratio, the encapsulation efficiency reached up to 80%. The encapsulation efficiency is determined as the weight percentage of cargo molecules forming polymer-cargo complexes with the biodynamer. Thus, encapsulation efficiency=weight of successfully complexed cargo molecules divided by total weight of cargo molecules.

[0173] Cell-uptake of polymer-cargo-complexes

[0174] Cellular uptake of the polymer-cargo-complexes formed from Lys-biodynamer and siRNA cargo molecules was tested using A549 cells.

[0175] The uptake increased with increased crosslinking ratio. By adding 50 w % of glutaraldehyde, the uptake increased to 84%. Thus, the polymer-cargo-complexes of the invention are suitable for delivering siRNA into cells. Differences in cell-uptake efficiency by NP ratio were not significant.

[0176] The results are shown in FIG. 9.