Bioconjugate Chem. 2004, 15, 831−840
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Cyclodextrin-Modified Polyethylenimine Polymers for Gene Delivery Suzie H. Pun,†,| Nathalie C. Bellocq,† Aijie Liu,† Greg Jensen,† Todd Machemer,‡ Erlinda Quijano,‡ Thomas Schluep,‡ Shufen Wen,‡ Heidrun Engler,‡ Jeremy Heidel,§ and Mark E. Davis*,§ Insert Therapeutics Inc., 2585 Nina Street, Pasadena, California 91107, Canji, Inc., 3525 Johns Hopkins Court, San Diego, California 92121, and Chemical Engineering, California Institute of Technology, Pasadena, California 91125. Received May 3, 2004; Revised Manuscript Received June 3, 2004
Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with β-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 µg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.
INTRODUCTION
Cationic polymers have received much attention for their potential use as nonviral gene delivery agents. Under certain conditions, these polymers self-assemble with poly(nucleic acids) via electrostatic interactions and condense them into submicron size particles that can be endocytosed by cells. Polymeric gene delivery vehicles have several advantages over their viral counterparts including nonimmunogenicity, adaptability, and ease of manufacture that make their development of interest for gene therapy applications. However, many polymers used for gene delivery are not suitable for systemic administration due to their toxicity and tendency to aggregate under physiological conditions (1). We have previously demonstrated the use of cyclodextrins (cyclic oligomers of glucose) as building blocks for the preparation of a suite of linear, cyclodextrin-based polycations (CDPs) that have been used for gene delivery (2-9). Like other polycations, these polymers assemble with plasmid DNA via electrostatic interactions to form small spherical particles with diameters ∼100 nm. The particles are able to transfect cultured mammalian cells and show low toxicity both in vitro and in vivo (2-9). The importance of the cyclodextrin in mediating low toxicity was demonstrated by the high in vitro toxicity of poly(amidine) polymers lacking cyclodextrins. Although these polymers still transfected cells with high efficiency, their IC50’s were 2-3 orders of magnitude lower than cyclodextrin-based poly(amidine) polymers (3, 5, 9). While other polycations show reduced toxicities with sugar and/or other functional group incorporation (10* To whom correspondence should be addressed. E-mail:
[email protected]. † Insert Therapeutics Inc. ‡ Canji, Inc. § California Institute of Technology. | Current address: Department of Bioengineering, University of Washington, Seattle, WA.
12), cyclodextrins are unique entities because they also serve as potential sites for modification via inclusion compound formation. Cyclodextrins are water-soluble, cup-shaped molecules that form inclusion complexes with small, hydrophobic compounds such as adamantane. The ability of cyclodextrins to form inclusion complexes can be utilized to modify the surface of the cyclodextrin-based, DNA-containing particles without interfering with polycation/DNA interactions and particle morphology (4, 8, 9). For example, adamantane-poly(ethylene glycol) (ADPEG) conjugates provide salt stability to β-cyclodextrinbased particles (4, 9). The adamantane in the AD-PEG conjugates form inclusion complexes with the β-cyclodextrins that are on the particle surfaces and thus anchor the PEG polymers to the surface to provide a PEG brush layer for particle stabilization. We have demonstrated the use of this inclusion formation technology to provide particle stability, tune particle surface charge, and modify particles with targeting ligands (9) such as galactose (4) and transferrin (8). This modification method utilizes self-assembly for the formulation, allows for great diversity in the types of surface modification, and is easily scalable. Poly(ethylenimine) (PEI) is a cationic polymer often used in nonviral gene transfer. PEI can provide for efficient, in vitro gene transfer, and this efficiency is speculated to be at least partially due to enhanced endosomal escape via pH-buffering (13, 14). PEI is one of the few polymers where transfection efficiency is not significantly increased by the presence of chloroquine, and this may be because of its mechanism of endosomal release (15). Nevertheless, the use of PEI as in vitro and in vivo transfection reagent is severely limited by its toxicity and difficulties in formulation. The LD50 of linear PEI (lPEI) is reported to be 4 mg/kg in Balb/C mice (16), 50-fold less than the LD50 of linear, cyclodextrin-based polymers (at least 200 mg/kg in Balb/C mice (3)). Particles formed from the condensation of PEI with poly(nucleic acids) (“PEI-based polyplexes”), like most polyplexes, also
10.1021/bc049891g CCC: $27.50 © 2004 American Chemical Society Published on Web 06/29/2004
832 Bioconjugate Chem., Vol. 15, No. 4, 2004
Pun et al.
Figure 1. (a) Synthesis of β-CD-bPEI. (b) Synthesis of β-CD-lPEI.
aggregate rapidly in physiological salt conditions. Approaches for improving the stability of PEI-based polyplexes include grafting of PEI with PEG and grafting of preformed PEI-based polyplexes with amine-reactive PEG (17, 18). However, PEI-PEG may not condense DNA into small spherical particles; rodlike and wormlike structures are often instead observed by transmission electron microscopy (1). Also, grafting of polyplexes with reactive PEG is difficult to control and scale-up. The objective of our study is to merge the beneficial qualities of the cyclodextrin-based polymers (low toxicity and the ability to modify the polyplex via inclusion complex formation) with the desirable features of the PEI polymers (efficient transfection). In this work, we describe the synthesis of β-cyclodextrin-grafted PEI (CD-PEI) polymers and their use as in vitro and in vivo nonviral gene delivery media. It is shown here that the transfection and toxicity profiles of the β-CD-PEI polymers are sensitive to the level of cyclodextrin grafting. The resulting polyplexes can be modified and stabilized by adamantane-poly(ethylene glycol) conjugates via inclusion complex formation. This modification creates particles that are formulated at high concentrations without flocculation, are stable in physiological salt concentrations, and are applicable for in vivo, systemic administration. Systemic delivery of plasmids containing the p53 gene with cyclodextrin-containing PEI delivery vehicles show significantly reduced toxicity when compared to PEI-based delivery vehicles in mice. Additionally, p53 gene expression is observed in liver when using cyclodextrin-containing PEI’s (unmodified PEI’s typically reveal expression in lung).
Table 1. Effect of Reaction Solvent on β-Cyclodextrin Grafting to BPEI H2O/DMSO (mL/mL)
amount of water (%)
amine grafting (%)
60/40 40/60 20/80 5/95 1/99 0.1/99.9 0/100
60 40 20 5 1 0.1 0
5 6 7 8 10 12 16
EXPERIMENTAL PROCEDURES
Synthesis and Characterization of CyclodextrinPEI Polymers. Preparation of Cyclodextrin-Grafted Branched PEI (CD-bPEI) (Figure 1a). Branched PEI25 000 (282 mg, 11.3 µmol, Aldrich, Milwaukee, WI) and 6-monotosyl-β-cyclodextrin (2.18 g, 1.69 mmol, Cyclodextrin Technologies Development, Inc.) were dissolved in 100 mL of various H2O/DMSO solvent mixtures (Table 1). The resulting solution was stirred at 70 °C for 72 h and then transferred to a Spectra/Por MWCO 10 000 membrane and dialyzed against water for 6 days. The dialyzed solutions were lyophilized to afford a slightly colored solid. Cyclodextrin/PEI ratio was calculated based on the proton integration of 1H NMR and shown in Table 1 (Varian 300 MHz, D2O) δ 5.08 ppm (s br., C1H of CD), 3.3-4.1 ppm (m br. C2HC6H of CD), 2.5-3.2 ppm (m br. CH2 of PEI). Preparation of Cyclodextrin-Grafted Linear PEI (CDlPEI) (Figure 1b). Linear PEI25 000 (500 mg, 20 µmol, Polysciences, Inc., Warrington, PA) and 6-monotosyl-βcyclodextrin (3.9 g, 3 mmol, Cyclodextrin Technologies
Cyclodextrin-Modified Polyethylenimine Polymers
Development, Inc.) were dissolved in 36 mL of DMSO. The resulting mixture was stirred at 70 °C for 6 days. The solution turned slightly yellow. The solution was then transferred to a Spectra/Por MWCO 10 000 membrane and dialyzed against water for 6 days. Water was then removed by lyophilization to afford a slightly colored solid. Cyclodextrin/PEI ratio was calculated based on the proton integration of 1H NMR (Varian 300 MHz, D2O) δ 5.08 ppm (s br., C1H of CD), 3.3-4.1 ppm (m br. C2HC6H of CD), 2.5-3.2 ppm (m br. CH2 of PEI) and found to be 1 CD: 8.4 amines. pH-Titration. Each polymer was dissolved to a concentration of 11.7 mM of amines in 150 mM NaCl and titrated with 0.1 N HCl or 0.1 N NaOH (19). pH measurements were carried out at 25 °C with a Beckman Coulter pH meter Φ340. Adamantane-Poly(ethylene glycol) (AD-PEG) Synthesis. AD-PEG was synthesized as described previously (4). In brief, PEG5000-SPA (Nektar AL, Huntsville, AL) was reacted with 5 equiv of 1-adamantanemethylamine (Aldrich) in dichloromethane. The solvent was removed in vacuo and water added to the remaining solution. The solution was centrifuged at 15 K rcf for 15 min to precipitate unreacted 1-adamantanemethylamine. The aqueous portion was then dialyzed (Slide-A-Lyzer MWCO 3500 Pierce, Rockford, IL) for 24 h against water and lyophilized to yield a white, fluffy powder in 97% yield. Cell Culture and Plasmids. PC3 (human prostatic carcinoma) cells were purchased from the ATCC and cultured in DMEM/F12 media containing 10% (v/v) fetal bovine serum containing 100 units/mL penicillin in a 5% CO2 humidified atmosphere at 37 °C. The pGL3-CV (containing the luciferase gene under the control of the SV40 promoter) and pEGFPLuc (containing the gene coding for an EGFP/luciferase fusion protein under the control of the CMV promoter) plasmids were purchased from Promega (Madison, WI) and Clontech (Palo Alto, CA), respectively, and amplified by Elim Biopharmaceuticals (Hayward, CA). Formulation and Characterization of CD-PEI/ DNA Complexes. Formulation. CD-PEI/DNA polyplexes were prepared by adding an equal volume of CDPEI solution (CD-PEI dissolved in dH2O) to a plasmid DNA solution (0.1 mg DNA/mL in dH2O) at the appropriate N/P ratio. PEGylated CD-PEI/DNA polyplexes were prepared by mixing equal volumes (25% of final volume) of CD-PEI and AD-PEG solutions (both dissolved in dH2O). The CD-PEI/AD-PEG solution was then added to an equal volume of plasmid DNA solution (0.1 mg DNA/mL in dH2O). Transmission Electron Microscopy. Polyplexes containing the pGL3-CV plasmid were prepared as described above. Five microliters of sample was then applied to glow-discharged, 400-mesh carbon-coated copper grids for 45 s, after which excess liquid was removed by blotting. The samples were then negatively stained with 2% uranyl acetate for 45 s before observation. Images were recorded using a Philips 201 electron microscope operated at 80 kV. Salt Stability Study. Two micrograms of pGL3-CV plasmid was formulated with CD-PEI, CD-PEI/ADPEG, PEI, or PEI/AD-PEG as described above. One sample was diluted by the addition of 1.2 mL of water to determine initial particle size. A second duplicate sample was then diluted by the addition of PBS (pH 7.2) to determine salt stability. Particle size was recorded every minute for 10 min using a ZetaPALS dynamic light
Bioconjugate Chem., Vol. 15, No. 4, 2004 833
scattering detector (DLS, Brookhaven Instruments Corporation, Holtsville, NY). In Vitro Transfection and Toxicity Assays. Luciferase Expression. PC3 cells (human prostate epithelial adenocarcinoma cells from ATCC) were plated at 50 000 cells/well in 24-well plates 24 h before transfection. Immediately prior to transfection, cells in each well were rinsed once with PBS before the addition of 200 µL of Opti-MEM (Invitrogen, Carlsbad, CA) containing polyplexes (1 µg of DNA complexed with polycation at 10 N/P). After 4 h, transfection media was aspirated and replaced with 1 mL of complete media. Forty-eight hours after transfection cells were washed with PBS and lysed by the addition of 100 µL of Cell Culture Lysis Buffer (Promega, Madison, WI). Cell lysates were analyzed for luciferase activity with Promega’s luciferase assay reagent. Light units were integrated over 10 s with a luminometer (Monolight 3010C, Becton Dickinson). Toxicity. The IC50’s of the various polymers to PC3 cells were determined by MTT assay as described previously (3). All experiments were conducted in triplicate and averaged. Average absorbance was plotted versus polymer concentration and IC50 values were determined by interpolation. Plasmid Delivery. PC3 cells were plated at 1 000 000 cells/well in six-well plates. After 24 h, the cells were exposed to 5 µg of fluorescently labeled plasmid (pEGFPLuc plasmid labeled with YOYO-1 (Molecular Probes, Eugene, OR) at a density of 1 dye molecule per 100 bp DNA) complexed with polymer at 10 N/P in 1 mL of OptiMEM (Gibco, Carlsbad, CA). Fifteen minutes after initial exposure, the polyplex-containing, culture media was aspirated. Cells were washed with PBS, Cell Scrub buffer (Gene Therapy Systems, San Diego, CA), trypsinized, and analyzed by flow cytometry for the uptake of the polyplexes. EGFP Expression. PC3 cells were plated at 250 000 cells/well in six-well plates. After 48 h, the cells were transfected with 5 µg of pEGFPLuc plasmid assembled with polymer at 10 N/P in 1 mL of OptiMEM (for some samples, OptiMEM containing 100 µM chloroquine was added). Four hours after transfection, the culture media was removed and replaced with 5 mL of complete media. Cells were washed with PBS, trypsinized, and analyzed by flow cytometry for EGFP expression 48 h after transfection. In Vivo Experiments. Tolerability was evaluated in immune competent female Balb/c mice. Animals were injected with the various formulations into the tail vein using a 0.4 mL volume and an injection speed of approximately 0.2 mL/15 s. Animals were sacrificed at 24 h after treatment by carbon dioxide euthanasia. Blood was collected by heart-puncture into sodium citrate containing tubes (platelets and white blood cell (WBC) counts) and serum tubes (transaminases, creatinine). Platelets and WBC counts were evaluated at Biomedical Testing Services Inc., and transaminases (ALT/AST) and creatinine using a Cobas Mira (Roche) autoanalyzer. Livers and lungs were collected, formalin fixed, sections H & E (hematoxilin (stains nucleus) and eosin (stains cytoplasm)) stained, and pathologically examined. Tolerability and transgene expression were evaluated in immune-compromised female nude mice bearing subcutaneous PC3 tumors. Tumors were established by injecting 1 × 107 PC3 cells in a volume of 0.2 mL subcutaneously in the left flank of animals. Tumors were grown to an average size of 100 mm3 before treatment. Animals were injected with complexes intravenously into the tail vein using a 0.4 mL volume and an injection
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speed of approximately 0.2 mL/15 s. Animals were sacrificed at 24 h after treatment and blood and serum collected as described above. Organs were collected into identically sized aluminum foil, weighed, and snap-frozen in liquid nitrogen for PCR/RT-PCR analysis. Quantitative PCR and RT-PCR (QPCR, QRT-PCR) were used to quantify p53 DNA and transgene expression as previously described (20). Briefly, DNA and RNA were coextracted from approximately 100 mg of tissue using Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH). Purified RNA was DNased and complete removal of DNA was confirmed by PCR. QPCR/QRT-PCR was performed using the ABI 7900 sequence detector (Applied Biosystems, Inc., Foster City, CA). Rodent GAPDH DNA and RNA were used as internal controls to assess the quality of purified RNA and DNA and to ensure equal amounts of sample input. Primer and probe sequence used for QPCR and QRT-PCR were as follows: p53; forward primer 5′-AACGGTACTCCGCCACC-3′, reverse primer 5′-CGTGTCACCGTCGTGGA-3′ and probe FAMCAGCTGCTCGAGAGGTTTTCCGATCC-TAMRA. GAPDH; forward primer, 5′-ACGTGCCGCCTGGAGAA-3′, reverse primer, 5′-CATGAGGTCCACCACCCTGTT-3′ and probe FAM-ATGACATCAAGAAGGTGGTGAAGCAGGCTAMRA. Tissue matched p53 standards were used to quantify p53 DNA levels in tissues as described previously (21). To generate the standardards, briefly, liver, kidney, lung and tumor were collected from naı¨ve animals. Tissues were minced thoroughly and aliquoted into several 100 mg samples. Six to eight 10-fold serially diluted samples of p53 plasmid, ranging from 1.0 × 103 to 1 × 109 particles, were spiked into each aliquot. p53-spiked tissue aliquots were extracted and processed for QPCR in a manner identical to the test samples, generating standard curves for each type of tissue. Standard curves were used to estimate the number of p53 DNA in test samples, which were expressed as genome equivalent fg per mg of tissue. p53 mRNA was quantified using rAd-p53 cRNA as reported previously (20) and was expressed as copies/ mg of tissue. RESULTS
Synthesis and Characterization of CD-bPEI and CD-lPEI Conjugates. β-Cyclodextrin-grafted poly(ethylenimines) (CD-PEI’s) were synthesized by reaction of mono-tosylated cyclodextrins with the primary and secondary amines of poly(ethylenimine) using a protocol similar to that described by Suh et al. (Figure 1) (22). This synthesis approach resulted in cyclodextrin conjugation to both branched and linear PEI (denoted here as CD-bPEI and CD-lPEI, respectively). The degree of CD substitution was determined by integration of the appropriate 1H NMR spectra and was found to depend on the amount of water in the reaction solvent. This effect was expected since the tosyl activating group is readily hydrolyzed by water. A series of CD-bPEI polymers with varying degrees of CD substitution (ranging from 5% to 16% amine substitution) were synthesized by changing the water to DMSO solvent ratio (Table 1). The transfection efficiency and toxicity of these various polymers were measured as described below to determine the synthesis conditions necessary for the best in vitro gene delivery behavior. CD-lPEI was also prepared following a similar protocol. Effect of CD-Grafting on Transfection and Toxicity of bPEI. The CD-bPEI polymers were formulated with pGL3-CV at charge ratios ranging from 2 N/P
Pun et al.
Figure 2. Effect of cyclodextrin grafting on CD-bPEI transfection efficiency to PC3 cells. CD-bPEI polymers were complexed with pGL3-CV plasmid at 10 N/P to form polyplexes for transfection. Luciferase activity was measured 48 h after transfection and reported as mean RLU/well ( SD for three replicates. Error bar on 0% CD grafting is too small to be observed.
(amine to phosphate) to 20 N/P and exposed to PC-3 cells plated in 24-well plates. The cells were lysed and analyzed for gene expression 48 h after transfection. Luciferase expression was detected at 4 N/P for all polymers and increased with charge ratio until toxicity was observed (generally around 15 N/P; results not shown). A comparison of transfection efficiency at 10 N/P as a function of CD grafting is shown in Figure 2. Luciferase activity is not normalized by total cell protein levels (RLU/mg) because this practice gives artificially high values for samples with low protein levels resulting from polymer toxicity. Instead, total luciferase activity per sample (RLU/well) is reported with polymer toxicity analyzed separately. Grafting of CD to bPEI reduces transfection efficiency. Modification of only 5% of the bPEI amines decreases luciferase activity by 1 order of magnitude (2 × 108 RLU/ well for bPEI vs (1-2) × 107 RLU/well for CD-bPEI with 5% to 8% amine grafting). CD grafting level is correlated with lower transfection; 10% amine modification results in a 2 orders of magnitude decrease in transfection to 1 × 106 RLU/well, and 16% amine modification reduces luciferase activity by over 4 orders of magnitude to 5 × 103 RLU/well. The toxicities of bPEI and the CD-bPEI polymers were determined by comparing the IC50 values of the polymers with PC3 cells as measured by the MTT assay (Figure 3). PC3 cells were plated in 96-well plates and exposed to serially diluted concentrations of the polymers. The cells were then assayed for cell viability 24 h after initial polymer addition and IC50 values calculated by interpolation. All IC50 values are reported as ethylenimine monomer concentrations. The IC50 values of CD-bPEI polymers are proportional to the extent of cyclodextrin grafting. At lower grafting percentages, polymer IC50’s are 2- to 5-fold higher (CD-bPEI IC50 of 0.64 mM and 1.45 mM for 5% and 8%, respectively, compared with bPEI’s IC50 of 0.28 mM). High levels of cyclodextrin grafting increases IC50 values by over 20-fold (4.8 mM for 12% grafting and 6.7 mM for 16% grafting). On the basis of the transfection and toxicity data, the CD-bPEI with 8% amine modification was chosen as the polymer with the best in vitro transfection and tolerability characteristics. This CD-bPEI polymer was used in the experiments described below. A similar synthesis procedure was used to prepare CD-lPEI (with 12% CD
Cyclodextrin-Modified Polyethylenimine Polymers
Bioconjugate Chem., Vol. 15, No. 4, 2004 835 Table 2. Blood Chemistry Data nude mice blood parameter D5Wa ALT (U/L)c AST (U/L)d Balb/c mice platelets (/mL)e WBC (/mL)f CRE (mg/dL) g
Balb/c mice
CD-lPEI + CD-lPEI + AD-PEGb untreated AD-PEGb
38 60
32 53
25 45
29 75
1175 12.5 n/a
1458 9.6 n/a
1335 7.4 0.8
624 6.4 0.9
a D5W: 5 wt % dextrose solution used as control. b Particles formulated with CD-lPEI + AD-PEG and plasmid DNA (120 µg). Average particle diameter was 117 nm. c Alanine aminotransferase (ALT). d Aspartate aminotransferase (AST). e Platelet count per milliliter. f White blood cell count per milliliter. g Creatinine.
Figure 3. Effect of cyclodextrin grafting on CD-bPEI toxicity to PC3 cells. Polymers were exposed to PC3 cells in serial dilutions for 24 h. Cells were then assayed for viability by MTT assay. The IC50’s, reported as ethylenimine monomer concentrations, were determined by interpolation on a best-fit line and reported mean values ( SD for three replicates.
Figure 4. Acid/base titration curves for bPEI, lPEI, CD-bPEI, and CD-lPEI polymers. The value reported on the x-axis is the change in concentration of hydrogen ion required to achieve the pH indicated on the y-axis. (a) bPEI and CD-bPEI; (b) lPEI and CD-lPEI.
modification). Cyclodextrin modification of lPEI reduced polymer toxicity. The IC50 for lPEI was increased by severalfold by cyclodextrin grafting (0.38 mM for lPEI compared with 0.92 mM for CD-lPEI; all IC50’s determined in triplicate and averaged). This CD-lPEI was also used in subsequent experiments. The titration curves of the CD-bPEI (8% amine modification), CD-lPEI (12% amine modification), and the unmodified bPEI and lPEI polymers are shown in Figure 4. Salt Stabilization of CD-PEI Polymers by ADPEG. CD-bPEI and CD-lPEI polymers self-assemble with and condense plasmid DNA to small spherical particles with average diameters ranging from 100 to 160 nm (not shown), as measured by dynamic light scattering (DLS). The particles can be PEGylated by the addition of AD-PEG5000 (poly(ethylene glycol) conjugated to adamantane). The adamantanes form inclusion complexes with the cyclodextrins on the surface of the particles,
resulting in particle PEGylation (Figures 5a and 5b). PEGylation does not significantly affect particle size or particle morphology (Figures 5c and 5d). The PEGylated particles, like the unmodified particles, are predominately spherical in structure. Salt stability of the unmodified and PEGylated CDPEI particles was tested by formulating the particles in a small volume of water (40 µL), adding 1.2 mL of PBS (150 mM salt concentration), and immediately monitoring average particle size by DLS with recorded measurements every minute for 10 min. All samples were run in triplicate and averaged for each time point. Figure 6 shows particle diameter versus time in salt solution for each type of formulation. All unmodified polyplexes aggregate in salt solutions. The addition of AD-PEG at 1 AD to 1 CD ratio reduces aggregation rate by forming a protective hydrophilic PEG brush layer on the particle surface. Complete particle stabilization is obtained by formulation with AD-PEG at 2 AD:1 CD (not shown). AD-PEG addition to bPEI and lPEI polyplexes does not stabilize the particles. Plasmid Uptake with CD-PEI Polymers. CDbPEI, CD-lPEI, bPEI, and lPEI were complexed with fluorescently labeled plasmids at 10 N/P and exposed to PC-3 (human prostatic carcinoma) cells for 15 min. The cells were then washed with PBS and Cell Scrub Buffer (to remove surface-associated polyplexes), trypsinized, and analyzed for polyplex uptake by flow cytometry. Uncomplexed plasmids were not readily taken up by cells (
