Throughput Design of Polyamidoamine Nanoparticles for Target Therapies

SUMMARYTriblock pH-responsive co-polymers were synthetized byRAFT polymerization and assembled in vesicles for thedelivery of siRNA to cancer cells. The copolymers self-assemble in spherical shape vesicles at pH 7.4 with a size of 60 nm, high homogeneity and good stability at 37°C.The carrier loads double stranded DNA sequences with a14% molar loading capacity and release the payload in 8hours at pH 5 while at pH 7.4 the release was negligible.The vesicles possess remarkable hemolytic activity at pH5 which can be correlated with potential endosome escapeof the system. Polymeric vesicles decorated with folateshowed a selective cell association with a model cancercell line overexpressing the folate receptor. INTRODUCTIONIn the last decades a variety of supramolecularsystems obtained by self-assembly of functional polymershave been developed with the aim of preserving thetherapeutic activity of loaded drugs whilst reducing theirside effects. However, these nanocarriers lack targetingmodalities and the ability to perform sequential operationsneeded to achieve controlled release of the drug at thebiological target. These limitations can be overcome by using nanosystems with both biorecognition capacity andthe responsive behavior to peculiar environmental stimuli.The aim of this study was to synthesize andinvestigate pH responsive triblock copolymers able toassemble into targeted vesicles for siRNA delivery tocancer cells. Folic acid was chosen to confer activetargeting for the biorecognition of folic acid receptor over-expressing tumor cells 1. Once endocytosed by thetarget cells, the pH responsive vesicles can disassemble inthe endosomes by protonation of ionizable monomers ofthe co-polymers and release the siRNA. EXPERIMENTAL METHODSSynthesis of triblock copolymer mPEG1900-poly(C6-imidazoyl-methacrylate-co-gliceroyl-methacrylate) (1)The co-polymer was synthetized by a three stepsprocedure: a) mPEG-OH was reacted with 4-cyano-4(phenylcarbonothioylthio)-pentanoic acid (CPAD), in thepresence of DMAP and DCC to obtaine a macro-transferagent (macro-CTA); b)C6-imidazoyl-methacrylate waspolymerized using the macro-CTA according to a RAFTpolymerization procedure; 3) mPEG-poly(C6-imidazoyl-methacrylate) was then used to polymerize glyceroyl-methacrylate by RAFT polymerization (see Scheme 1 forchemical structure).Synthesis of t-Boc–NH–PEG3500-poly(C6-imidazoylmethacrylate-co-gliceroyl-methacrylate) (2). The co-polymer was synthetized as reported for (1) using t-Boc-NH-PEG-OH as starting material.Synthesis of folate–NH–PEG3500-poly(C6-imidazoylmethacrylate-cogliceroyl-methacrylate) (3). Polymer (2)was treated with a 1:1 (v/v) CF3COOH/DCM mixture toremove the Boc agent; the unprotected polymer wasisolated by solvent evaporation and reacted with N-hydroxysuccinimidyl ester activated folic acid in DMSO.The crude product was purified by dialysis using a 3.5kDa cut-off dialysis bag (see Scheme 1 for structure). Scheme 1. Chemical structure of triblock copolymers (1) on the left and (3) on the right. All polymers were characterized by1H-NMR, GPC, potentiometric titration, turbidimetric and PhotonCorrelation Spectroscopic (PCS) analysis.Vesicles assembly. Targeted vesicles were assembledusing a 90:5:5 w/w ratio of polymers (1)/(2)/(3). Controluntargeted vesicles were prepared with a 90:10 w/w ratioof polymer (1)/(2). The vesicles were assembled bydissolving 1 mg/ml of the copolymers in PBS at pH 3 andincreasing the pH to 7.4 using 0.1 M NaOH. The vesiclesformation was confirmed by PCS.Kinetic stability studies. The stability of 1 mg/ml vesicles was performed by analyzing the size evolutionover time with PCS. The investigation was carried out inPBS at pH 7.4 at 37°C with and without foetal bovinserum.DNA loading and release. A Tris buffer solutions of19-mer oligomeric dsDNA (100 μM) was added topolymer mixtures in PBS at pH 4 to achieve a 10:1 and1:1 N/P ratio. Polymer mixtures had the samecomposition reported above to obtain targeted and untargeted vesicles. Polymers were then induced toassemble by the pH increase method. The dsDNA loadingyield and release from the nanocarrier was at 37°C at pH7.4 and 5.0 by Uv-Vis spectroscopy.TEM images. dsDNA-free and dsDNA-loadedpolymersomes were analyzed by TEM in negativestaining mode and the contrast staining was performedwith a 1% w/v uranyl acetate solution.CN