Novel bioreducible poly(amido amine)s for highly efficient gene delivery

A series of novel bioreducible poly(amido amine)s containing multiple disulfide linkages (SS-PAAs) were synthesized and evaluated as nonviral gene vectors. These linear SS-PAAs could be easily obtained by Michael-type polyaddition of various primary amines to the disulfide-containing cystamine bisacrylamide. The SS-PAA polymers are relatively stable in medium mimicking physiological conditions (pH 7.4, 150 mM PBS, 37 degrees C), but are rapidly degraded in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment (pH 7.4, [R-SH] = 5 mM, 37 degrees C). The polymers efficiently condense DNA into nanoscaled (<200 nm) and positively charged (>+20 mV) polyplexes that are stable under neutral conditions but are rapidly destabilized in a reductive environment, as was revealed by both dynamic light scatting measurement and agarose gel assays. Moreover, most of the poly(amido amine)s possess buffer capacities in the pH range pH 7.4-5.1 that are even higher than polyethylenimine (pEI), a property that may favorably contribute to the endosomal escape of the polyplexes. Polyplexes of four of the seven SS-PAAs studied were able to transfect COS-7 cells in vitro with transfection efficiencies significantly higher than those of branched pEI, being one of the most effective polymeric gene carriers reported to date. Importantly, also in the presence of serum, a high level of gene expression could be observed when the incubation time was elongated from 1 h to 4 h. XTT assays showed that SS-PAAs and their polyplexes possess essentially no or only very low cytotoxicity at concentrations where the highest transfection activity is observed. The results indicate that bioreducible poly(amido amine)s have excellent properties for the development of highly potent and nontoxic polymeric gene carriers.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

Temperature-sensitive pluronic/poly(ethylenimine) nanocapsules for thermally triggered disruption of intracellular endosomal compartment

Pluronic hydrogel nanoparticles cross-linked with poly(ethylenimine) (PEI) were synthesized by a modified emulsification/solvent evaporation method. Pluronic F-127 preactivated at the terminal group with p-nitrophenyl chloroformate was dissolved in dichloromethane, and the organic solution was emulsified in deionized water containing PEI by sonication. Primary amine groups of PEI in the aqueous phase were conjugated and/or cross-linked with activated Pluronic F-127 in the vicinity of the water/dichloromethane interface, resulting in the formation of shell-cross-linked Pluronic/PEI nanocapsules. Pluronic/PEI nanocapsules exhibited a volume transition behavior over a temperature range of 24-33 degrees C. The thermally reversible swelling/deswelling of Pluronic/PEI nanocapsules was caused by temperature-dependent hydrophobic interaction of cross-linked and/or grafted Pluronic polymer chains in the nanocapsules. Pluronic/PEI nanocapsules were utilized to break up intracellular endosomal compartments by swelling-induced destabilization of the endosomal membrane triggered by a cold-shock treatment.     

Ruthenium(II) tris(bipyridine)-centered poly(ethylenimine) for gene delivery

Ruthenium(II) tris(bipyridine)-centered poly(ethylenimine) (Ru PEI) was synthesized via acid hydrolysis of Ru tris(bipyridine)-centered poly(2-ethyl-2-oxazoline) (Ru PEOX), and the luminescence, DNA entrapment, and transfection efficiencies were evaluated. Emission maxima for Ru PEI samples are red-shifted compared to Ru PEOX precursors, and the luminescence lifetimes are shorter in both methanol and aqueous solutions. Slower oxygen quenching of Ru PEOX and Ru PEI luminescence versus [Ru(bpy)3]Cl2 (bpy = bipyridine) is attributed to polymer shielding effects. Ru PEI luminescence is similar in the presence and absence of DNA. Ru PEI (7900 Da) and linear PEI (L-PEI; 22,000 Da) fully entrapped DNA (5.4 kb; pcDNA) at an N/P ratio of 2. LNCaP prostate cancer cells were transfected with a plasmid encoding for green fluorescent protein using Ru PEI and L-PEI vectors for comparison. For N/P = 48, the transfection efficiency for Ru PEI was approximately 50% relative to that of L-PEI.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

Amphiphilic core-shell nanoparticles with poly(ethylenimine) shells as potential gene delivery carriers

Spherical, well-defined core-shell nanoparticles that consist of poly(methyl methacrylate) (PMMA) cores and branched poly(ethylenimine) shells (PEI) were synthesized via a graft copolymerization of methyl methacrylate from branched PEI induced by a small amount of tert-butyl hydroperoxide. The PMMA-PEI core-shell nanoparticles were between 130 to170 nm in diameter and displayed zeta-potentials near +40 mV at pH 7 in 1 mM aqueous NaCl. Plasmid DNA (pDNA) was mixed with nanoparticles and formed complexes of approximately 120 nm in diameter and was highly monodispersed. The complexes were characterized with respect to their particle size, zeta-potential, surface morphology, and DNA integrity. The complexing ability of the nanoparticles was strongly dependent on the molecular weight of the PEI and the thickness of the PEI shells. The stability of the complexes was influenced by the loading ratio of the pDNA and the nanoparticles. The condensed pDNA in the complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxity studies using MTT colorimetric assays suggested that the PMMA-PEI (25 kDa) core-shell nanoparticles were three times less toxic than the branched PEI (25 kDa). Their transfection efficiencies were also significantly higher. Thus, the PEI-based core-shell nanoparticles show considerable potential as carriers for gene delivery.     

Engineering a polymeric gene delivery vector based on poly(ethylenimine) and hyaluronic acid

In this work, the effects of primary amines, ligand targeting, and overall charge on the effectiveness of branched poly(ethylenimine)-hyaluronic acid conjugate (bPEI-HA) zwitterionic gene delivery vectors are investigated. To elucidate the relative importance of each of these parameters, we explored the zeta potential, cytotoxicity, and transfection efficiency for a variety of formulations of bPEI-HA. It was found that the length of the hyaluronic acid (HA) oligosaccharide had the most significant effect on cytotoxicity and transfection efficiency with human mesenchymal stem cells. Test groups of bPEI incorporating HA with a length of 10 saccharides had significantly higher transfection efficiency (14.6 ± 2.0%) and lower cytotoxicity than other formulations tested, with the cytotoxicity of the group containing the greatest mass of 10 saccharide showing similar results as the positive controls at the highest polymer concentration (100 μg/mL). Additionally, molar incorporation of HA, as opposed to the saccharide length and HA mass incorporation, had the greatest effect on zeta potential but a minor effect on both cytotoxicity and transfection efficiency. This work demonstrates the relative importance of each of these tunable design criteria when creating a zwitterionic polymeric gene delivery vector and provides useful specific information regarding the design of bPEI-HA gene delivery vectors.     

Dual bio-responsive gene delivery via reducible poly(amido amine) and survivin-inducible plasmid DNA

A bioreducible poly(amido amine) (SS-PAA) gene carrier, known as poly (amido-butanol) (pABOL), was used to transfect a variety of cancer and non-cancer cell lines. To obtain cancer-specific transgene expression for therapeutic efficiency in cancer treatment, we constructed survivin-inducible plasmid DNA expressing the soluble VEGF receptor, sFlt-1, downstream of the survivin promoter (pSUR-sFlt-1). Cancer-specific expression of sFlt-1 was observed in the mouse renal carcinoma (RENCA) cell line. pABOL enhanced the efficiency of gene delivery compared to traditional carriers used in the past. Thus, a dual bio-responsive gene delivery system was developed by using bioreducible p(ABOL) for enhanced intracellular gene delivery and survivin-inducible gene expression system (pSUR-sFlt-1 or pSUR-Luc reporter gene) that demonstrates increased gene expression in cancer that has advantages over current gene delivery systems.     

Synthesis of poly(beta-amino ester)s optimized for highly effective gene delivery

Several families of synthetic polymers, including degradable poly(beta-amino ester)s, have been previously shown to effectively mediate gene transfer. However, the combined impact of potentially significant factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on different gene transfer properties has yet to be systematically investigated. The elucidation of these relationships may aid in the design of nonviral vectors with greatly enhanced transfection properties. To examine these factors, two distinct poly(beta-amino ester) structures, Poly-1 and Poly-2, were generated by adding 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively, to 1-aminobutanol. Twelve unique versions of each structure were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in polymers with either amine or acrylate end-groups and with molecular weights ranging from 3350 to 18000. Using high throughput methods, all polymers were tested in quadruplicate at nine different polymer/DNA ratios ranging from 10:1 w/w to 150:1 w/w. Through the optimization of molecular weight, polymer chain end-group, and polymer/DNA ratio, these polymers successfully mediated gene transfer at levels that surpassed both PEI and Lipofectamine 2000 in vitro.     

Visualization of the degradation of a disulfide polymer, linear poly(ethylenimine sulfide), for gene delivery

Polyethylenimine (PEI) shows high transfection efficiency and cytoxicity due to its high amine density. The new disulfide cationic polymer, linear poly(ethylenimine sulfide) (l-PEIS), was synthesized for efficient and safe gene delivery. As the amine density of l-PEIS increased, the transfection efficiency also increased. l-PEIS-6 and l-PEIS-8 show transfection efficiencies that are similar to that of PEI. However, cytotoxicity of l-PEIS was not observed due to the biodegradable disulfide bond. The disulfide bonds are stable in the oxidative extracellular condition and can be degraded rapidly in the reductive intracellular condition. The degradation of l-PEIS in HeLa cells was visualized by fluorescence microscopy using the probe-probe dequenching effect of BODIPY-FL fluorescence dye. l-PEIS was degraded completely within 3 h.     

Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(L-aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (K(SV)). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t(1/2)) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.     

pH-responsive poly(styrene-alt-maleic anhydride) alkylamide copolymers for intracellular drug delivery

Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.     

A tetra(L-lysine)-grafted poly(organophosphazene) for gene delivery

In order to develop a new gene delivery vector, a novel cationic poly(organophosphazene) was synthesized by stepwise nucleophilic substitutions of poly(dichlorophosphazene) with a hydrophilic methoxy-poly(ethylene glycol) (MPEG) as a shielding group and a branched tetra(L-lysine), LysLys(LysEt)(2), as a cationic moiety. The cationic polymer has shown to form a polyplex by DNA condensation and very low in vitro cytotoxicity probably due to the shielding effect of MPEG, which provides a basis for improving the low gene transfection yield of cationic polyphosphazenes.     

Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.     

Effects of metal ions on the hydrolysis of p-nitrophenyl caproate by modified poly(ethylenimine)s

Hydrolysis of nitrophenyl caproate by modified poly(ethylenimine)s containing imidazole moieties is markedly enhanced in the presence of divalent metal ions. The order of effectiveness is Cu(II) > Co(II) > Zn(II) > Ni(II) > Mn(II), with Cu increasing the rate about 20-fold. The acceleration by the metal ion is much greater in the presence of CH₃COO^? or Cl^? as counterions than in the presence of ClO₄^?. Turnover experiments, in which moles of substrate cleaved were in substantial excess of moles of metal present, established the catalytic nature of the effects of the metals. The extent of binding of Cu(II) by the polymers was measured. Maximum accentuation in rate of hydrolysis of nitrophenyl ester was observed for the imidazole-containing polymer when the ratio of imidazole:Cu(II) was 2.75. Some possible mechanisms for the rate enhancement by metal ions are described.     

Gene delivery properties of end-modified poly(beta-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.     

Dendritic poly(ether imine) based gene delivery vector

The nonviral vector based gene delivery approach is attractive due to advantages associated with molecular-level modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether imine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.     

Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide)

End-functionalized poly(N-isopropylacrylamide) (PNIPA) was synthesized by living free radical polymerization and conventional free radical polymerization and was used to prepare graft copolymers with poly(ethylenimine) (PEI). The copolymers exhibited lower critical solution temperature (LCST) behavior between 30 and 32 degrees C and formed complexes with plasmid DNA. The LCST of the copolymers in the DNA complexes increased slightly to approximately 34-35 degrees C. Cytotoxicity of the copolymers was evaluated by measuring lactate dehydrogenase (LDH) release from cells. The copolymers exhibited temperature-dependent toxicity, with higher levels of LDH release observed at temperatures above the LCST. Cellular uptake and transfection activity of the DNA complexes with the PEI-g-PNIPA copolymers were lower than those of the control PEI/DNA complexes at temperature below the LCST but increased to the PEI/DNA levels at temperatures above the LCST.     

Structural advantage of dendritic poly(L-lysine) for gene delivery into cells

This study aimed to investigate the relationships between structures of gene carrier molecules and their activities for gene delivery into cells. We compared 2 types of poly(L-lysine) as carriers, that is, dendritic poly(L-lysine) (KG6) and linear poly(L-lysine) (PLL). KG6 formed a neutral DNA complex, and its DNA compaction level was weaker than that of PLL. The amount of DNA binding and uptake into cells mediated by PLL was 4-fold higher than that with KG6. However, KG6-mediated gene expression was 100-fold higher than that by PLL. Since pK(a) values of terminal amines of KG6 were lowered even though small amounts of DNA were internalized into cells, sufficient DNA amounts for effective gene expression escaped to the cytosol due to the proton sponge effect in the endosome. In addition, weakly compacted DNA with KG6 was advantageous in accessing RNA polymerase in the cell nucleus. On the other hand, PLL did not show the proton sponge effect in the endosome and resulted in strong compaction of DNA. Even though large DNA amounts were internalized into cells, most of the DNA would not take part in gene expression systems in the nucleus. Amount of induced cytokine production after intravenous injection of DNA complexes with KG6 and PLL was low, and was similar to the case when DNA was injected alone. Therefore, no significant difference in effects on cytokine production was observed between KG6 and PLL.     

Self-aggregates of oligoarginine-conjugated poly(amino acid) derivatives as a carrier for intracellular drug delivery

N_ω-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (N_ω-Pbf)-protected oligoarginine was directly conjugated to poly(amino acid) derivatives modified with a long alkyl chain. The final concentration of conjugated peptides was easily controlled by the feed ratio of oligoarginine to polymer backbone and a final soluble polymeric system was obtained by the deprotection of N_ω-Pbf groups. The polymeric conjugates formed stable self-aggregates of size range of 8–40 nm in aqueous solution and effectively internalized into HeLa cells by adsorptive endocytosis.Revisions requested 8 April 2005; Revisions received 6 May 2005