Poly-l-glutamic Acid Derivatives as Multifunctional Vectors for Gene

Sep 25, 2003 - as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stabi...
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Biomacromolecules 2003, 4, 1177-1183

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Poly-L-glutamic Acid Derivatives as Multifunctional Vectors for Gene Delivery. Part B. Biological Evaluation Peter Dubruel,† Luc Dekie,† Bart Christiaens,‡ Berlinda Vanloo,‡,§ Maryvonne Rosseneu,‡ Joe¨l Vandekerckhove,§ Marjo Mannisto,| Arto Urtti,| and Etienne Schacht*,†,⊥ Polymer Materials Research Group, Department of Organic Chemistry, Ghent University, Ghent, Belgium, Laboratory of Lipoprotein Chemistry, Department of Biochemistry, Ghent University, Ghent, Belgium, Flanders Interuniversity Institute for Biotechnology (VIB), Department of Medical Protein Research, Ghent University, Ghent, Belgium, Department of Pharmaceutics, University of Kuopio, Kuopio, Finland, and Institute Biomedical Technology, Ghent University (IBITECH), Ghent, Belgium Received January 13, 2003; Revised Manuscript Received May 16, 2003

Cationic polymers, such as poly-L-lysine (pLL) and polyethyleneimine (pEI), are receiving growing attention as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stability toward nucleases. The major disadvantages of using both polymers for in vivo purposes are their cytotoxicity and, in the case of pEI, the fact that it’s not biodegradable. In this work, we investigated the interaction between a series of cationic, glutamic acid based polymers and red blood cells. The MTT test was used to investigate the cytotoxicity of the complexes. The ability of the polymers to stabilize DNA toward nucleases was investigated. Transfection studies were carried out on Cos-1 cells. The results from the haemolysis studies, the haemagglutination studies, and the MTT assay show that the polymers are substantially less toxic than pLL and pEI. The polymers are able to protect the DNA from digestion by DNase I. The transfection studies show that the polymer-DNA complexes are capable of transfecting cells, most of them with poor efficiency compared to pEI-DNA complexes. 1. Introduction Biotechnology has evolved to the point that it is possible to prepare therapeutic genes for most diseases. The use of these new medicines is hampered by the lack of a suitable carrier system that helps the DNA in reaching the target cells and in the subsequent intracellular trafficking. Cationic polymers have been proposed as vectors for genetic material because they readily form polyelectrolyte complexes with DNA.1-9 A drawback of using basic polymers such as pLL and pEI is that they interact with erythrocytes, hereby causing haemolysis and haemagglutination.10-12 Katchalsky showed earlier that the interaction is electrostatic in nature and depends on the structure of the polymer used.13 Furthermore, polymers such as pEI are not biodegradable which means that repetitive administration of this vector can cause lysosomal storage disease.14-16 To avoid this problem, we synthesized a new series of cationic polymers based on glutamic acid that are biodegradable by lysosomal enzymes.17 The synthesis and the physicochemical evaluation of these polymers was studied in a separate paper in the same issue of this journal.18 This study showed that most polymers were able to condense DNA, resulting in a reduced DNA surface * To whom correspondence should be addressed. Prof. Dr. E. H. Schacht, Krijgslaan 281 (S4 Bis), B-9000 Ghent, Belgium. Phone: 0032-(09)2644497. Fax: 0032-(0)9-2644998. E-mail: [email protected]. † Department of Organic Chemistry, Ghent University. ‡ Department of Biochemistry, Ghent University. § Department of Medical Protein Research, Ghent University. | University of Kuopio. ⊥ Institute Biomedical Technology, Ghent University.

charge and particle size. In addition, the stability of the polymer-DNA complexes toward serum albumin and the buffering properties of the different polymers was studied. The stability studies showed that the different polymer-DNA complexes are stable (do not dissociate) upon incubation with serum albumin at physiological concentrations (50 g/L). The titration studies showed that the polymers have buffering properties in the pH range covering the endosomal compartment. To investigate a possible effect of these findings on the biological properties of the polymer-DNA complexes, we have carried out an extensive biological evaluation of the complexes including haemolysis studies, agglutination studies and MTT tests. We also investigated the stability of the DNA present in the complexes toward nucleases (DNase I). Finally, transfection studies were performed on Cos-1 cells. 2. Materials and Methods 2.1. Chemicals. Bovine serum albumin (Sigma), Lipofectamine (Gibco-BRL), branched polyethyleneimine (Aldrich) Mw ) 25 000 Da, highly polymerized calf thymus DNA (CT DNA, Fluka), DNase I (type IV from bovine pancreas, Sigma), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, Aldrich), and tris(hydroxymethyl)aminomethane (Tris, Avocado) were used as such. The glutamic acid based polymers were prepared as previously described.18 The bicinchoninic acid kit for protein determination was purchased from Pierce. The β-Gal ELISA kit for measuring the β-Gal mass was obtained from Roche. A 8.3 kb

10.1021/bm034015b CCC: $25.00 © 2003 American Chemical Society Published on Web 07/10/2003

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expression vector containing CMV promotor-driven β-galactosidase, produced in E. coli, was used for assessing gene expression during the transfection experiments. The endotoxin-free plasmid Maxiprep kit was obtained from Qiagen. 2.2. Polymer-DNA Complex Preparation. For calculation of the charge ratio, a mass per charge of 325 was used for DNA. The mass per charge of the polymer was calculated assuming 100% protonation of the primary amines, tertiary amines, imidazole, and guanidine functions (Table 1). These assumptions are not completely correct, but they allow us to consistently estimate the mass per charge of the polymers. Each sample, except those used in the transfection studies, contained 20 µg/mL of CT DNA and the appropriate polymer in the desired charge ratio (total sample volume was 2 mL). The complexes were allowed to self-assemble in water and were left at room temperature for at least half an hour before use. 2.3. Red Blood Cell Preparation. Bovine blood was used throughout the experiments. Erythrocytes were washed three times at 4 °C by centrifugation at 4000 rpm and resuspension in HBS buffer (20 mM HEPES, 150 mM NaCl, pH ) 7.4). A 2% stock suspension was prepared by suspending the cells after the third washing in the HBS buffer and stored at 4 °C. 2.4. Interaction with Erythrocytes. 2.4.1. Haemolysis Study. In these experiments, the interactions between both the free polymers and the complexes and red blood cells were evaluated. The various polymers were added to the red blood cells in a concentration range of 1-2000 µg/mL. The complexes were prepared at a 0.5:1; 1:1, 2:1, and 4:1 (+:-) charge ratio and added to a 2% red blood cell suspension. The suspensions were incubated for 24 h at 37 °C. The supernatants were spinned off at 4000 rpm for 5 min. Haemolysis was investigated and quantified by measuring the haemoglobin release at 545 nm. Triton X-100 (10%) and HBS were used to provide the 100% and 0% haemolysis values, respectively. The % haemolysis is calculated as follows: % lysis )

Apolymer/complex - Ablank 100% Atriton - Ablank

with Ablank ) absorbance of a red blood cell suspension in absence of polymer or polymer-DNA complex Atriton ) absorbance in the presence of Triton X-100 (10%) Apolymer/complex ) absorbance in the presence of polymer or polymer-DNA complex 2.4.2. Agglutination Study. The ability of the polymers and polymer-DNA complexes to agglutinate red blood cells was investigated by means of optical microscopy using Nomarsky illumination. Complexes were added in a 2:1 (+:-) charge ratio to a 2% red blood cell suspension. Free polymer was added to the erythrocytes in the same quantity as present in the complexes. The suspensions were incubated for at least 1 h at 37°C. Samples (5 µl) were put on a glass plate and a picture was taken (630× magnification).

Dubruel et al. Table 1. Mass Per Charge of the Different Cationic Polymers polymer

mass/charge (Da)

pDMAEG pDMAEG85%-pAEG15% pDMAEG82%-pHisG18% pDMAEG64%-pHisG36% pDMAEG35%-pHisG65% pDMAEG24%-pHisG76% pDMAEG12%-pHisG88% pHisG pDMAEG84%-pAgmG16% pHisG73%-pAgmG27% pEI

235 231 233 230 226 225 224 222 241 237 208

2.5. Cytotoxicity Study. Two cell lines were used: EA.hy 926 cells (real genuine endothelial cells) and D 407 cells (retinal epithelial cells). The cytotoxicity of the polymerDNA complexes was determined via the MTT assay. A detailed protocol was described earlier.19,20 The results are expressed as % decrease in MTT reduction of treated cells compared to the MTT reduction of untreated cells (100% cell viability). The equation used to calculate the % cell viability is given below: % viability )

Ameasured - Ablank × 100% Ahealthy cells - Ablank

with Ablank ) absorbance of a DNA solution Ameasured ) absorbance of cells + polymerDNA complexes Ahealthy cells ) absorbance of cells - polymerDNA complexes 2.6. Stability of DNA toward DNase I. The stability of the DNA present in the polyelectrolyte complexes toward digestion by nucleases was monitored using DNase I. The complexes were prepared in a Tris buffer (40 mM Tris-HCl, 10 mM NaCl, 6 mM MgCl2 and 10 mM CaCl2, pH ) 5) at a 0.2:1, 0.5:1, and 2:1 (+:-) charge ratio. 40 units of DNase I (1 unit/µg DNA) were added, and the degradation was followed at 25 °C in function of time by measuring the increase in absorbance at 260 nm. A sample containing only DNA was used as control. The complexes were not destabilized before performing OD measurements. 2.7. Transfection of Cos-1 Cells. 2.7.1. Plasmid Preparation. The plasmid was expanded in DH10B E. coli and was isolated using the Qiagen endotoxin-free plasmid Maxiprep kit, according to the supplier’s protocol. The quality and the quantity of the purified plasmid DNA was assessed by spectrophotometric analysis at 260 and 280 nm, as well as by electrophoresis on a 1% agarose gel. Purified DNA was resuspended in water and frozen (-20 °C) in aliquots at a concentration of 0.1 mg/mL. 2.7.2. Transfection Protocol. The monkey kidney fibroblast Cos-1 cell line was grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (104 IU/mL), further

Part B. Biological Evaluation

referred to as complete medium. The transfection protocol used for pEI and the poly-L-glutamic acid based DNA carriers is based on an available protocol for Lipofectamine. The day before transfection, Cos-1 cells were seeded at a density of 2 × 104 cells/cm2 in 12-well plates and were grown for 24 h in complete medium. Two hours before transfection, the cells were washed twice with DMEM and incubated for 2 h in DMEM (400 µL/well, 37 °C, 5% CO2). The transfection mixtures, containing the polymer-DNA complexes, were prepared as follows. Three compounds were added and mixed in a precise order: DNA, water, and polymer. The final volume of the mixtures was 80 µL, containing 0.06 mg/mL plasmid DNA and the appropriate amount of polymer to obtain the desired charge ratio. Immediately after addition of the polymer, the mixtures were vortexed for 30 s and further incubated for 2 h at room temperature. pEI and Lipofectamine were used as reference material. The amount of Lipofectamine used was 37 µg, according to the protocol of the supplier. Then, 13 µL transfection mixture was added to each well (780 ng DNA/well). The cells were incubated for 4 h with the transfection mixtures (37 °C, 5% CO2). After this incubation, the cells were washed twice with DMEM and incubated for 48 h in 1 mL complete medium (37 °C, 5% CO2). Finally, the cells were washed three times with cooled PBS (4 °C), lysed with 1 mL lysis buffer, and harvested. The quantification of the β-gal mass was performed using the β-gal ELISA kit, according to the supplier’s protocol. Cellular protein determinations were performed with the BCA Protein Assay kit.

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Figure 1. Haemoglobin release by the free polymers after 24 h incubation at 37 °C. The polymers were incubated at different concentrations with 2% red blood cell suspensions. The degree of haemolysis was quantified by OD measurements at 545 nm.

3. Results and Discussion 3.1. Biocompatibility Studies. In in ViVo gene therapy, a vector system is brought into the bloodstream of patients. It is therefore of the utmost importance that the vectors are biocompatible and show a minimal interaction with blood components. The research on biocompatibility is very complex and is related to the interactions that occur between polymers or polymer-DNA complexes and cells. Therefore, we investigated the interaction of the different polymers and polymer-DNA complexes with red blood cells by haemolysis and haemagglutination studies. The toxicity was tested on cells in culture. 3.1.1. Blood Interaction Studies. The red blood cell membrane consists of 43% phospholipids, 49% proteins, and 8% carbohydrates.21,22 They can be either bound to proteins or to lipids. The membrane of erythrocytes is negatively charged due to the great amount of neuramic acids present at the extracellular side of the cell. Cationic polymers are capable of lysing and agglutinating red blood cells.23 This interaction is due to the high charge density of the polyion and occurs at the surface of the cell. 3.1.1.1. Haemolysis Studies. Haemolysis is caused by destabilization of the erythrocyte membrane by free polycations or polymer-DNA complexes. This is monitored by measuring the release of haemoglobin (absorbance measurements at 545 nm). The results for the free polymers are shown in Figure 1. It can be seen that the polyglutamic acid

Figure 2. Haemoglobin release by polymer-DNA complexes after 24 h incubation at 37 °C. The complexes were prepared at different charge ratios and incubated with 2% red blood cell suspensions. The degree of haemolysis was quantified by OD measurements at 545 nm.

based polymers are less haemotoxic than pEI and pLL. pDMAEG64%-pHisG36% is less haemotoxic than pDMAEG. This is probably due to the lower charge density of this polymer. The imidazole groups have a pKa of 7 and are therefore not fully protonated (charged) under the experimental conditions (neutral pH). The lower haemotoxicity of pDMAEG compared to pLL is due to the different chemical structure of the charged groups. Most likely, the two methyl groups of the tertiary amines cause steric hindrance, resulting in a weaker interaction with the membrane components. Comparing these results with those from erythrocytes lysis caused by polymer-DNA complexes, it can be seen that there is a reduced haemotoxicity for complexes compared to the free polymers (see Figure 2). The complexes based on pLL and pEI possess the highest haemotoxicity of all complexes tested. This is due to the lower charge or zeta potential of the complexes based on pDMAEG, pHisG, and pDMAEG64%-pHisG36% compared to those based on pLL and pEI.18 3.1.1.2. Haemagglutination Studies. Polycations agglutinate erythrocytes within minutes and at very low concentra-

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Figure 3. Agglutination of erytrocytes by cationic polymers and polymer-DNA complexes. Complexes were prepared at a 2:1 (+:-) charge ratio.

tions (few µg/mL).13,24 This agglutination is due to the polymeric character because cationic monomers do not cause agglutination, even at high concentrations (5 mg/mL). The same applies for neutral or anionic polymers. PLL and polyvinylamine cause agglutination in concentrations ranging from 1 to 2 µg/mL after 3-5 min incubation, whereas protamine and polyvinyl piperidine agglutinate red blood cells at concentrations of 60 and 100 µg/mL, respectively.13 The results of the agglutination studies for the different polymers are shown in Figure 3. Free pLL causes severe agglutination forming aggregates having sizes of up to 300 µm. Similar results were obtained for pEI (data not shown). pDMAEG causes less agglutination. The polymer induces some aggregation forming clusters of cells with sizes up to 40 µm. pDMAEG82%-pHisG18% and pHisG cause almost no agglutination. In some cases, clusters are seen consisting of up to 20 cells, but in most cases, individual cells are spotted. pLL and pEI based complexes are less toxic than the free polymers but still agglutinate red blood cells forming aggregates with sizes up to 150 µm. This is shown for pLLDNA complexes in Figure 3. Complexes based on pD-

MAEG, pDMAEG82%-pHisG18% and pHisG induce practically no aggregation. It can be concluded from these results that primary amines have a pronounced toxic effect on red blood cells. Furthermore, we can say that, because of a lower surface charge, complexes cause less agglutination than the free polymer. These results are of the utmost importance for a possible in vivo application of these gene delivery systems. 3.1.2. Cytotoxicity Study. The cell viability can be evaluated by means of the MTT test. The results are given in Figure 4. It can be concluded that complexes containing the multifunctional glutamic acid derivatives are much less toxic than those formed with pEI. As in the haemolysis and haemagglutination studies, this is probably due to the more pronounced interaction of pEI with the cell membrane which results in membrane rupture and cell death. Only pHisG73%pAgmG27% shows a toxicity which is higher compared to the other glutamic acid based derivatives. 3.2. Stability of DNA toward Nucleases. Nucleases are omnipresent in the body and are necessary for cellular and viral development. Nucleases are involved in the protective mechanisms against foreign DNA, the degradation of host

Part B. Biological Evaluation

Figure 4. Cytotoxicity of polymer-DNA complexes as determined by the MTT assay. The compounds were evaluated both in EA.hy 926 cells (part A) and D 407 cells (part B). Complexes were prepared at different charge ratios.

DNA after viral infection, DNA repair, DNA synthesis, etc. Rapid degradation occurs when free DNA is brought into contact with nucleases.25,26 The DNA is protected from the digestive action of the enzymes when it is condensed. The extent to which the DNA is protected depends on the type of vector system used.27,28

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The results for complexes prepared at a 0.2:1, 0.5:1, and 2:1 (+:-) charge ratio. The degradation was followed after addition of the nucleases using UV spectroscopy at 260 nm.29 The results are shown in Figure 5. pLL based complexes show the largest DNA degradation at a low charge ratio. There is almost complete protection at a 2:1 (+:-) charge ratio. pDMAEG based complexes give a better protection toward DNase I. There is some slight degradation of the DNA present in the complexes prepared at a 0.5:1 (+:-) charge ratio. The DNA in the complexes formed at a 2:1 (+:-) charge ratio is fully protected from the digestive action of the enzymes. When the complexes based on pHisG and pDMAEG64%pHisG36% are compared, it can be seen that those containing pDMAEG64%-pHisG36% give the best protection toward DNase I. Although pHisG was not able to condense the DNA,18 we see that the polymer does protect it toward nucleases. The reason for this is unknown. Maybe the polymer gives some steric hindrance so that the enzyme cannot properly bind to the substrate. 3.3. Transfection Studies. The ultimate goal of gene therapy is the introduction and expression of a therapeutic gene by a specific cell type. Therefore, we tested the ability of our polymers to transfect Cos-1 cells in culture. Lipofectamine and pEI were used as controls during these experiments. From the results shown in Figure 6, one can conclude that the complexes, except those based on pHisG73%-pAgmG27%, transfect Cos-1 cells with poor efficiency. The low efficiency is probably due to the fact that the complexes are not able to properly interact with the membrane of the cells as was shown from the biocompatibility studies and, thus, are not taken up into the cells. The pHisG73%-pAgmG27% based complexes, prepared at a 8:1 (+:-) charge ratio, give a transfection efficiency that is higher than that of pEI-DNA

Figure 5. Degradation of DNA present in various polymer-DNA complexes by DNAse I. The complexes were prepared in a Tris buffer at a 0.2:1, 0.5:1, and 2:1 (+:-) charge ratio. 40 units DNase I (1 unit/µg DNA) were added and the degradation was followed as a function of time by measuring the increase in absorbance at 260 nm, A (260 nm). A sample containing only DNA was used as control.

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Figure 6. Transfection of polymer-DNA complexes in Cos-1 cells. The results are expressed as picogram β-galactosidase (pg β-gal) per mL of solution. Cos-1 cells were seeded at a density of 2 × 104 cells/cm2. 13 µL transfection mixture was added to each well (780 ng DNA/well). The cells were incubated for 4 h with the transfection mixtures. (A) pDMAEG:DNA, (B) pDMAEG64%-pHisG36%:DNA, (C) pDMAEG24%-pHisG76%: DNA, (D) pDMAEG12%-pHisG88%:DNA, (E) pHisG:DNA, (F) pHisG73%-pAgmG27%:DNA, (G) pEI:DNA.

Figure 7. Amount of cell protein for the different complexes studied in Cos-1 cells. The results are expressed as microgram cell protein per ml solution (µg/mL). (A) pDMAEG:DNA, (B) pDMAEG64%-pHisG36%:DNA, (C) pDMAEG24%-pHisG76%:DNA, (D) pDMAEG12%-pHisG88%:DNA, (E) pHisG:DNA, (F) pHisG73%-pAgmG27%:DNA, (G) pEI:DNA.

complexes prepared at a 2:1 (+:-) charge ratio. The exact reason why these complexes give such good results is not yet understood. It may be due to the presence of the guanidine functions. The chemical structure of the guanidine side groups from the polymers and the amino acid arginine are quite similar. It is well-known that arginine rich peptides, such as the human immunodeficiency virus (HIV-1) Tat(48-60) and Drosophila Antennapedia (pAntp)-(43-58) (penetratin-1) are taken up into cells very efficiently.30 The transfection data presented in this work might be an indication that the positively charged guanidine function is an essential component in the translocation mechanisms of arginine rich peptides. The amount of cell protein for the different complexes is presented in Figure 7. The results indicate that Lipofectamine and pEI are very toxic to cells. The amount of cell protein for the glutamic acid based complexes is between 80% and 100% of untreated cells (cells treated with free DNA). These results indicate that complexes that are not able to transfect

Cos-1 cells are not cytotoxic. Cells treated with pHisG73%pAgmG27% based complexes at a 8/1 charge ratio have an amount of cell protein which is 63% of untreated cells. These results confirm those of the MTT assay in EA.hy 926 cells and D 407 cells where it was observed that pHisG73%pAgmG27%-DNA complexes are more toxic compared to the other glutamic acid based polymers. Future work in this field will be directed to the study of the cellular uptake and the intracellular distribution of polymer-DNA complexes. Because of their promising transfection results, the emphasis will be on polymers containing agmatine groups. Polymers containing fluorescent groups have been prepared and are currently being tested out using confocal microscopy. The results will be reported in a forthcoming paper. 4. Conclusions The investigated polymers and polymer-DNA complexes cause much less haemo- and cytotoxicity than pLL or pEI

Part B. Biological Evaluation

and their corresponding complexes. As was shown, this is probably due to the different chemical structure of the cationic groups and the reduced zeta potential of the complexes. Results from the nuclease degradation studies showed that the polymers were able to protect the DNA from the digestive function of DNase I. The majority of complexes were unable to transfect Cos-1 cell culture. Only the complexes based on pHisG73%pAgmG27% were able to transfect the cells to a similar extent as pEI-DNA complexes. Future work will be done to optimize the structure of the polymers (e.g., targeting groups) so that the transfection efficiency can be improved. Incorporation of fluorescent dyes will allow us to further elucidate the interaction between the complexes and the cells. Acknowledgment. The authors thank the Flemish institute for the promotion of Scientific-Technological Research in Industry (IWT), the Fund for Scientific Research-Flanders (FWO), the Belgian Ministry of Scientific Programming, IUAP/PAI-V, and the European Union’s Biotechnology Program Contract Number 97 2334 with support from INCO Contract Number IC 20 CT 970005. We are thankful to Prof. W. Vyverman for the use of their microscope and K. Vanhoutte for his guidance while using the apparatus. The group of X. Van Ostade is greatly acknowledged for supplying us with the E. coli and for the electroporation. Abbreviations pLL: poly(L-lysine) pEI: polyethyleneimine pDMAEG: poly(dimethylaminoethyl-L-glutamine) pDMAEG-pAEG: poly(dimethylaminoethyl-L-glutamine)-copoly(aminoethyl-L-glutamine) pDMAEG-pHisG: poly(dimethylaminoethyl-L-glutamine)-copoly(histamino-L-glutamine) pHisG: poly(histamino-L-glutamine) pDMAEG-pAgmG: poly(dimethylamino-ethyl-L-glutamine)co-poly(agmatino-L-glutamine) pHisG-pAgmG: poly(histamino-L-gluta-mine)-co-poly(agmatino-L-glutamine) CT DNA: calf thymus DNA DNase: deoxyribonuclease HBS: HEPES buffered saline PBS: phosphate buffered saline TRIS: tris(hydroxymethyl)aminomethane HEPES: N-(2-hydroxyethyl)piperazine-N′-(2-ethane-sulfonic acid) SDS: sodium dodecyl sulfate.

Note Added after Print Publication. The spelling of the author name Joe¨l Vandekerckhove was incorrect in the version published on the Web on 7/10/2003 (ASAP) and in print (Biomacromolecules 2003, 5, 1177-1183). The correct

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electronic version of the manuscript was published on 9/25/ 2003, and an Addition and Correction appears in the November/December 2003 issue (Vol. 4, No. 6). References and Notes (1) Shapiro, J. T.; Leng, M.; Felsenfield, G. Biochemistry 1969, 8 (8), 3219-3232. (2) Wu, G. Y.; Wu, C. H. J. Biol. Chem. 1987, 262 (10) 4429-4432. (3) Kawai, S.; Nishizawa, M. Mol. Cell. Biol. 1984, 4:6, 1172-1174. (4) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A. Angew. Chem.-Int. Ed. Engl. 1990, 29 (2), 138-175. (5) Kabanov, A. V.; Astafieva, I. V.; Chikindas, M. L.; Rosenblat, G. F.; Kiselev, V. I.; Severin, E. S.; Kabanov, V. A. Biopolymers 1991, 31, 1437-1443. (6) van de Wetering, P.; Cherng, J. Y.; Talsma, H.; Crommelin, D. J. A.; Hennink, W. E. J. Controlled Release 1998, 53 (1-3), 145-153. (7) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (16), 7297-7301. (8) Dubruel, P.; Toncheva, V.; Schacht, E. H. J. Bioact. Compat. Polym. 2000, 15 (3), 191-213. (9) Dubruel, P.; Schacht, E. H. J. Bioact. Compat. Polym. 2000, 15 (4), 279-296. (10) Nevo, A., De Vries, A., Katchalsky, A. Biochim. Biophys. Acta 1955, 17, 536-547. (11) De Vries, A.; Stein, Y., Stein, O.; Feldman, J.; Gurevitch, J.; Katchalsky, E. Proc. 4th Int. Congr. Int. Soc. Haematol. 1954, 385390. (12) Moreau, E.; Ferrari, I.; Drochon, A.; Chapon, P.; Vert, M.; Domurado, D. J. Controlled Release 2000, 64 (1-3), 115-128. (13) Katchalsky, A.; Danon, D.; Nevo, A. Biochim. Biophys. Acta 1959, 33, 120-138. (14) De Duve, C.; De Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P.; Van Hoof, F. Biochem. Pharmacol. 1974, 23, 2495-2497. (15) http://www.genzyme.com. (16) http://www.botresearch.com/concepts/part4.html. (17) Dekie, L.; Toncheva, V.; Dubruel, P.; Schacht, E. H.; Barrett, L.; Seymour, L. W. J. Controlled Release 2000, 65 (1-2), 187-202. (18) Dubruel, P.; Dekie, L.; Schacht, E. Biomacromolecules 2003, 4, 1168. (19) Mannisto, M.; Vanderkerken, S.; Toncheva, V.; Elomaa, M.; Ruponen, M.; Schacht, E.; Urtti, A. J. Controlled Release 2002, 169189. (20) Hyvonen, Z.; Plotniece, A.; Riene, I.; Chekavichus, B.; Duburs, G.; Urtti, A. Biochim. Biophys. Acta 2000, 1509 (1-2), 451-466. (21) Cantor, R.; Schimmel, P. R. In Biophysical Chemistry Part 1; W. H. Freeman and Company: New York, 1999; p 235. (22) Ferruti, P.; Manzoni, S.; Richardson, S. C. W.; Duncan, R.; Pattrick, N. G.; Mendichi, R.; Casolaro, M. Macromolecules 2000, 33:21, 7793-7800. (23) Moreau, E.; Ferrari, I.; Drochon, A.; Chapon, P.; Vert, M.; Domurado, D. J. Controlled Release 2000, 64 1-3, 115-128. (24) Rubini, J. R.; Stahmann, M. A.; Rasmussen, A. F., Jr. Proc. Soc. Biol. Med. 1951, 76, 659-662. (25) Wickstrom, E. J. Biochem. Biophys. Methods 1986, 13:2, 97-102. (26) Liu, G.; Molas, M.; Grossmann, G. A.; Pasumarthy, M.; Perales, J. C.; Cooper, M. J.; Hanson, R. W. J. Biol. Chem. 2001, 276 (37), 34379-34387. (27) Chiou, H. C.; Tangco, M. V.; Levine, S. M.; Robertson, D.: Kormis, K.; Wu, C. H.; Wu, G. Y. Nucleic Acids Res. 1994, 22 (24), 54395446. (28) Gao, X.; Huang, L. Biochemistry 1996, 35:3, 1027-1036. (29) Kunitz, M. J. Gen. Physiol. 1950, 33, 349-361. (30) Futaki, S. Int. J. Pharm. 2002, 245, 1-7.

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