Surface Modification of Microspheres with Steric Stabilizing and

School of Pharmacy, UniVersity of Nottingham, UniVersity Park, Nottingham NG7 2RD, and AstraZeneca. R&D Charnwood, Bakewell Road, Loughborough ...
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Langmuir 2008, 24, 7138-7146

Surface Modification of Microspheres with Steric Stabilizing and Cationic Polymers for Gene Delivery Owen R. Davies, Laura Head, David Armitage, Elizabeth A. Pearson, Martin C. Davies, Maria Marlow, and Snjezana Stolnik* School of Pharmacy, UniVersity of Nottingham, UniVersity Park, Nottingham NG7 2RD, and AstraZeneca R&D Charnwood, Bakewell Road, Loughborough LE12 5RH, United Kingdom ReceiVed NoVember 29, 2007. ReVised Manuscript ReceiVed March 20, 2008 In this paper, we describe surface modification of poly(D,L-lactide-co-glycolide) (PLG) microspheres, intended for DNA vaccine application, with two functionalities: a steric stabilizing component, provided by poly(vinyl alcohol) (PVA) and a cationic component, aimed at subsequent DNA surface loading. The cationic functionality arises from polycations, such as PEI, poly(L-lysine), trimethyl chitosan, and (dimethylamino)ethyl methacrylate, introduced into the water phase of classical oil-in-water (o/w) solvent evaporation method of PLG microsphere fabrication. By systematic evaluation of production variables, a system was produced with balanced properties in terms of microsphere size appropriate for uptake by antigen presenting (e.g., dendritic) cells, colloidal stability, and relatively high DNA loading. The polycation (PEI) molecular weight and preparation concentration were both found to increase the surface polycation content and DNA binding capacity; however, they lead to an increased tendency for aggregation, particularly when the microsphere size was decreased. DNA loading of almost 100% efficiency was achieved under optimized conditions in physiologically acceptable buffers, resulting in a surface DNA loading appropriate for vaccine purposes. A further increase in surface DNA loading was however associated with an increase in the particles negative potential, indicating the surface presence of DNA charges not neutralized by the polycation and hence potentially not protected from in ViVo enzymatic degradation. The internalization of surface-loaded DNA into the target cells was confirmed by monitoring fluorescent DNA after the microspheres were endocytosed by the cells in culture.

Introduction Biodegradable polymeric microspheres have been extensively investigated as adjuvants for both protein and, more recently, DNA encoded antigens. These systems are able to protect entrapped or adsorbed molecules from degradation in ViVo and facilitate their uptake by professional antigen presenting cells responsible for generating adaptive immune responses. The most studied particulate vaccine delivery systems to date are those produced from the biodegradable, biocompatible polymer poly(D,Llactide-co-glycolide).1–7 DNA is typically entrapped within the microspheres using a double w/o/w emulsion technique.1–4,7 This procedure however exposes the DNA molecule to a number of stresses, for example, high shear forces and a liquid-liquid interface, which can lead to significant degradation of the DNA.1,7 Furthermore, due to its high molecular weight, entrapped DNA is released very slowly and, hence, may not achieve a desired therapeutic effect in addition to potentially being denatured due to the presence of acidic residues inside the microspheres’ matrix which are created as the PLG hydrolyzes.8 Consequently, it has * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 01158466074. Fax: 01159515102. (1) Capan, Y.; Woo, B. H.; Gebrekidan, S.; Ahmed, S.; DeLuca, P. P. Pharm. Res. 1999, 16(4), 509–513. (2) Chen, S. C.; Jones, D. H.; Fynan, E. F.; Farrar, G. H.; Clegg, J. C. S.; Greenberg, H. B.; Herrmann, J. E. J. Virol. 1998, 72(7), 5757–5761. (3) Jones, D. H.; Corris, S.; McDonald, S.; Clegg, J. C. S.; Farrar, G. H. Vaccine 1997, 15(8), 814–817. (4) McKeever, U.; Barman, S.; Hao, T.; Chambers, P.; Song, S.; Lunsford, L.; Hsu, Y. Y.; Roy, K.; Hedley, M. L. Vaccine 2002, 20(11-12), 1524–1531. (5) Singh, M.; Briones, M.; Ott, G.; O’Hagan, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97(2), 811–816. (6) Singh, M.; Vajdy, M.; Gardner, J.; Briones, M.; O’Hagan, D. Vaccine 2001, 20(3-4), 594–602. (7) Wang, D. Q.; Robinson, D. R.; Kwon, G. S.; Samuel, J. J. Controlled Release 1999, 57(1), 9–18. (8) Fu, K.; Pack, D. W.; Klibanov, A. M.; Langer, R. Pharm. Res. 2000, 17(1), 100–106.

been proposed that more potent immune responses may be evoked if the DNA is adsorbed onto preformed microspheres.5 This can be achieved by surface-modifying the microspheres to produce cationic particles to which DNA can be adsorbed through electrostatic interactions. Incorporation of cationic surfactants into the microsphere production was initially suggested;5,6,9 however, the toxicity of such materials needs to be taken into consideration. The second approach is based on the synthesis of PLG-polycations.10–12 For instance, particles have been produced from a poly(D,L-lactic acid)/poly(L-lysine) graft copolymer using emulsion and diafiltration techniques,11 from poly(-CBZ-Llysine)/poly(lactic-co-glycolic acid),10 and more recently, PLGPEI copolymer microspheres have been produced by spraydrying12 and precipitation techniques.13 Polycation-modified microspheres have been demonstrated to bind plasmid DNA to their surface and transfect cell lines in Vitro.10,12 In the present work, a classical o/w emulsion technique was modified to allow for simultaneous surface modification of PLG microspheres with a polycation, capable of binding DNA to particle surface, and poly(vinyl alcohol). The latter is an established material required as an emulsion stabilizer in the production of PLG microspheres and was, in the present work, aimed at providing steric stabilization to the final microsphere suspension, particularly once the charge stabilization arising from the polycation ceases to exist due to its neutralization by DNA. Systematic physicochemical characterization focused on optimizing a balance between, on one side, the effects that polycation (9) Cui, Z. R.; Mumper, R. J. J. Pharm. Pharmacol. 2002, 54(9), 1195–1203. (10) Manuel, W. S.; Zheng, J.; Hornsby, P. J. J. Drug Targeting 2001, 9(1), 15+ (11) Maruyama, A.; Ishihara, T.; Kim, J. S.; Kim, S. W.; Akaike, T. Bioconjugate Chem. 1997, 8(5), 735–742. (12) Walter, E.; Merkle, H. P. J. Drug Targeting 2002, 10(1), 11–21. (13) Bivas-Benita, M.; Romeijn, S.; Junginger, H. E.; Borchard, G. Eur. J. Pharm. Biopharm. 2004, 58(1), 1–6.

10.1021/la703735n CCC: $40.75  2008 American Chemical Society Published on Web 06/18/2008

Surface Modification of Microspheres

molecular weight, its surface content, and particle size have on the DNA loading and, on the other side, the PVA steric stabilization effects and colloidal stability of the microspheres: an approach that, to our knowledge, has not been investigated for such systems.

Experimental Section Materials. Poly(D,L-lactide-co-glycolide) (PLG) end-capped structure (50:50 ratio of lactide to glycolide, MW 40-75 000, PLG) and fluorescamine were purchased from Sigma, UK. RG502H, a PLG molecule with free terminal carboxyl groups (50:50 ratio of lactide to glycolide, MW 16 500 Da), was obtained from Boehringer Ingelheim, Germany. Green fluorescent protein (GFP) plasmid DNA was obtained from Aldevron, North Dakota, USA. Poly(vinyl alcohol) (MW 25 000, 88% hydrolyzed, PVA) was purchased from Fisher Scientific, Loughborough, UK. Polyethylenimine (PEI) branched (MW 25 000, branched MW 2000, and linear MW 400) and thiazole orange were obtained from Aldrich, Milwaukee, USA. All other chemicals were reagent-grade. Preparation of Microspheres. Cationic poly(D,L-lactide-coglycolide) microspheres were prepared using an o/w solvent evaporation method. A 10% (w/v) solution of poly(vinyl alcohol) in distilled water was prepared by overnight stirring. The obtained PVA solution was mixed with the polycation and diluted with distilled water to give a solution containing 5% (w/v) PVA and polyethylenimine (0.1-1% (w/v)). Large microspheres (average size 4 µm) were prepared by emulsifying a solution of PLG in dichloromethane (DCM, 10% (w/v), 1 mL) with the PVA/polycation mix (12 mL) for 5 min at 6000 rpm using a Silverson homogenizer (Silverson Machines Ltd., UK). Smaller microspheres (1.5-1.8 µm) were prepared by homogenizing the PLG solution (5% (w/v), 2 mL) with 20 mL of the PVA/polycation at 19 000 rpm for 5 min using an IKA Ultra-Turrax T25 homogenizer (IKA Werke, GmbH, Germany). Microspheres were left at room temperature in a fume hood for at least 4 h to allow the dichloromethane to evaporate and microparticles to harden. Microspheres were then recovered by centrifugation at 10 000 rpm, washed three times with distilled water, and freezedried for 24 h. Particle Size Distribution. A sample of 5 mg of PLG microspheres was dispersed in 0.5 mL of distilled water. The suspension was then added to the sample chamber of a Coulter LS 230 (Beckman Coulter Ltd., UK) under moderate stirring to the required concentration (as indicated by the display). Particle size was determined as a function of the particle diffraction using the Coulter software (version 2.11a). Particle size distribution was then plotted as a function of volume percentage. Microsphere Zeta Potential Measurement. Microsphere zeta potential was measured at room temperature in filtered 1 mM NaCl solution (adjusted to pH 7.4) using a Zetasizer 4 (Malvern Instruments, UK). Approximately 10 mL of microsphere suspension was transferred into a syringe and injected through the injection port of the instrument. After each injection, the capillary tube was flushed with 20 mL of filtered buffer solution. Determination of Plasmid DNA Loading. Microspheres were incubated with a known amount of DNA at room temperature for 1 h. Samples were rotated end over end in Eppendorf tubes to ensure that microspheres remained dispersed. At the end of the incubation period, samples were centrifuged at 8000 rpm for 3 min. The supernatant was then removed and diluted accordingly. A 100 µL aliquot was then mixed with 50 µL of thiazole orange solution (25 µg/mL) and the fluorescence read at 485/535 nm in an MRF plate reader (Dynex Technologies, UK). Unbound DNA content was analyzed by comparison to a standard curve, from which DNA loading was determined. Determination of Microsphere PEI Content. The PEI content of the microspheres was analyzed using a sensitive florescence method based on the fluorescamine protein assay.14 Microspheres (3 mg) (14) Weigele, M.; Debernar, S; Leimgrub, W Biochem. Biophys. Res. Commun. 1973, 50(2), 352–356.

Langmuir, Vol. 24, No. 14, 2008 7139 were weighed into Eppendorf tubes and dissolved in 300 µL dimethylsulfoxide (DMSO) by rotating end over end at room temperature. After approximately 1 h, samples were diluted accordingly, and a 100 µL aliquot was then mixed with 50 µL of fluorescamine dissolved in DMSO (0.3 mg/mL). After a 15 min incubation period at room temperature, the fluorescence was read at 355/460 nm in an MRF plate reader (Dynex Technologies, UK). Microsphere PEI content was then determined by comparison to a standard curve. Optical Microscopy. GFP plasmid DNA was adsorbed to the microspheres at a target DNA loading level of 3 µg DNA/mg microspheres. At the end of the 1 h incubation period, one drop of microsphere suspension was placed on a glass microscope slide. A glass coverslip was then placed over the droplet to minimize evaporation. Slides were examined under a Leica DMIRB inverted microscope (Leica, UK). Digital images were taken at 20-40× magnification with a Leica DC200 color video camera (Leica, UK) using the associated Leica DCViewa software (version 3.2.0.0., Germany). Further image processing was performed using QWin software (Leica, UK). Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Control spectra of individual materials were taken by drop-casting a 10 mg/mL solution (prepared in clean distilled water for PEI and PVA, and in chloroform for PLG) onto a silicon substrate. Microsphere samples were prepared by drop-casting a 10 mg/mL suspension of microspheres (prepared in clean distilled water) onto a silicon substrate to form a “raft” of microspheres. All samples were dried in air prior to analysis using an Ion-ToF time of flight secondary ion mass spectrometer IV Micro Analyzer. A gallium (15 KeV) primary ion source was used for both positive and negative spectra (Ion-ToF, Germany). All spectra were acquired while maintaining a primary ion dose of less than 1012 ions cm-2 in order to remain below the static limit. Emitted secondary ions were analyzed in terms of their mass/charge (m/z) ratio, yielding positive and negative SIMS spectra. The mass resolution at (m/z 41), defined as m/∆m, was just over 6000. Charge neutralization was achieved using lowenergy electron flux (20 eV). All ToF-SIMS spectra were calibrated using hydrocarbon fragment analysis. Microsphere Confocal Microscopy Studies. Confocal laser scanning microscopy (CLSM) was used to analyze microspheres for the presence of PEI 25 000 and adsorbed DNA. To show the presence of PEI 25 000 at the microsphere surface, microspheres were prepared as above except that the PEI in the aqueous phase was replaced by Oregon Green 488 carboxylic acid succinimidyl ester labeled PEI 25 000.15 To show the presence of DNA at the microsphere surface, YOYO-1 iodide-labeled GFP plasmid DNA was used (1 molecule of dye per 300 base pairs of nucleotide.16 Labeled DNA was adsorbed to the microspheres at a loading level of 1 µg/mg as described above. At the end of the incubation period, microspheres were washed three times to remove any loosely bound material. Microspheres were then examined using a Leica confocal system (TCS SP, Leica UK) attached to a Leica DNLFS microscope (Leica, UK). Oregon Green, coumarin-6, and YOYO-1 dyes were excited with the 488 nm argon laser line of the confocal microscope, and fluorescence was detected between 500 and 560 nm. A z-series of optical images was collected using a 63× oil emersion objective lens and displayed as the maximum intensity projection of all the images. These were generated using the associated software (Leica Confocal software version 2.5, USA). Cell Uptake Studies. Uptake of microspheres was assessed using the human monocyte cell line MonoMac 6. Cells were maintained as a suspension cell line in a humidified atmosphere at 37 °C, 5% (v/v) CO2. Cells were grown in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 1 mM sodium pyruvate, 9 µg/mL bovine insulin, and 1% (v/v) nonessential amino acids. (15) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96(9), 5177–5181. (16) Ogris, M.; Wagner, E.; Steinlein, P. Biochim. Biophys. Acta 2000, 1474(2), 237–243.

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DaVies et al. Table 1. Effect of PEI 25 000 Preparation Concentration on Resultant Microsphere Propertiesa sample

PEI contentb

zeta potentialc

control -8.5 ( 0.5 0.1% (w/v) PEI 25 000 1.31 ( 0.05 2.3 ( 0.5 0.5% (w/v) PEI 25 000 2.39 ( 0.04 4.7 ( 0.4 1.0% (w/v) PEI 25 000 2.44 ( 0.15 4.6 ( 0.2

DNA adsorption capacityd 0.17e 1.35 ( 0.09 2.46 ( 0.03 2.57 ( 0.17

a Results represent the mean ( 1 standard deviation of triplicate batches, with three samples per batch. b Expressed as µg PEI/mg microspheres. c Expressed in mV. d Expressed as µg DNA/mg microspheres (measured in 10 mM phosphate buffer, pH 7.4, at a loading level of 3 µg/mg microspheres). e Average of triplicate samples from one batch.

Figure 1. DNA loading capacity of microspheres modified with a range of cationic polymers at a cationic polymer concentration of 0.1% w/v and a PVA concentration of 5% w/v. LPEIslinear PEI MW 400 Da, TMCstrimethyl chitosan MW 116 000 Da, DMAs(dimethylamino)ethyl methacrylate MW 12 700 Da, PLLspoly(L-lysine) MW 30 000-70 000.

Cells were subcultured every three days with a split ratio of 1:3 to 1:5. A sterile glass microscope coverslip was placed in each well of a 6-well plate. An aliquot containing 1 × 105 cells in 1.5 mL of cell culture media was then added to each well followed by a further 1.5 mL of cell culture media containing 50 ng/mL of phorbol myristate acetate (PMA), to give a final PMA concentration of 25 ng/mL. The cells were incubated at 37 °C and 5% CO2. After 72 h, the media was removed and replaced with fresh media. Microsphere uptake prior to DNA adsorption was assessed using coumarin-6 labeled microspheres (0.1% (w/w), which were prepared by dissolving the dye in the organic phase (Desai et al., 1997). A suspension of microspheres was prepared by dispersing 4 mg of microspheres in 100 µL of sterile 0.9% saline. A volume of 10 µL of microsphere suspension was added to each well (200 µg). The cells were then returned to the incubator. After 24 h, the coverslip was carefully removed, rotated, and placed on a glass slide already mounted with a drop of PBS. Cells were then examined by confocal microscopy at 488 nm as described above. DNA uptake was assessed using YOYO-1-labeled DNA. A suspension of microspheres was prepared by dispersing 4 mg of microspheres in 150 µL of sterile 0.9% (w/v) saline. A volume of 50 µL (20 µg) of YOYO-1 iodide-labeled GFP plasmid DNA was then added. Microspheres were rotated end over end at 15 rpm at room temperature for 1 h in the dark. 10 µL (200 µg) of microsphere suspension was added to each well. Following an incubation period of 4 h at 37 °C, cells were prepared and analyzed using confocal microscopy as described above.

Results and Discussion PLG microspheres with an average size of 4.0 µm were prepared using an emulsification method in which both the steric stabilizing poly(vinyl alcohol) (PVA) and the polycation were codissolved in the water phase. An initial screening experiment was conducted to ascertain the generic nature of the approach (Figure 1). All the polycations were, at this stage of the work, used at the same initial preparation concentration, and the systems were not optimized for DNA loading. The main objective was to demonstrate that a wide range of polycations, including PEI, poly(L-lysine), trimethyl chitosan, and (dimethylamino)ethyl methacrylate could be incorporated at the microsphere surface and subsequently bind DNA. From this experiment, different molecular weights of PEI were selected for further systematic study.

Effect of PEI Concentration on Microsphere Surface Properties. The effect of PEI 25 000 presence in the aqueous phase on the properties of the microparticles is illustrated in Table 1. The content of PEI at the surface of the microspheres was determined using a florescence method based on the fluorescamine protein assay.14 The results demonstrate that, as the PEI concentration in the water phase was increased from 0.1% to 0.5% (w/v), the surface PEI content increased from 1.31 to 2.39 µg PEI/mg microspheres. A further increase in input concentration of PEI from 0.5% (w/v) to 1% (w/v) had no significant effect on the final content. This is likely to be a consequence of the particle surface becoming saturated with polycation where further accumulation is prevented due to intermolecular repulsion. The values for the microsphere zeta potentials showed charge inversion from negative values for control microspheres, prepared in the absence of PEI, to positive values for microparticles prepared in the presence of the polycation. The trend is in agreement with the increase in PEI content, clearly indicating that the positive charges are arising from PEI present at the surface. The maximum DNA adsorption capacity of the microspheres, under the stated conditions (10 mM phosphate buffer, pH 7.4, target DNA loading level of 3 µg DNA/mg microspheres), was approximately 2.5 µg DNA/mg microspheres. This value is comparable to PLG microsphere systems where the DNA has been encapsulated; values of 1.76 to 2.7 µg of DNA per mg of PLG or 3.4 to 4.5 µg DNA/mg lyophile.2,4 Furthermore, “washing” of microspheres did not result in any significant release of DNA, indicating its relatively stable binding to the microsphere surface. Confocal microscopy further confirms the presence of PEI 25 000 and DNA at the microparticle surface (Figure 2). No differences in surface morphology could be detected by scanning electron microscopy before and after DNA adsorption (Figure 2). The production of the PLG microspheres used in this work is based on the initial emulsification of an organic solvent phase, with dissolved PLG polymer, in an aqueous phase that contains PVA as stabilizer. Studies to understand the behavior of PVA at the liquid-liquid interface of the initial emulsion are scarce. The reported neutron reflectometry study indicated that PVA of MW 37 KDa, similar to the one used in the present study, forms a thin layer on the order of 1-2 nm thickness at the hexane/water interface, with PVA being positioned preferably in the water phase.27 Subsequent solidification of PLG during the microparticles production creates a solid-liquid interface, and evidence has been presented that PVA eventually becomes physically entrapped at the microsphere surface.17 It can be envisaged that (17) Scholes, P. D.; Coombes, A. G. A.; Illum, L.; Davis, S. S.; Watts, J. F.; Ustariz, C.; Vert, M.; Davies, M. C. J. Controlled Release 1999, 59(3), 261–278.

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Figure 2. Scanning electron micrographs and confocal images of PEI 25 000 modified microspheres before and after plasmid DNA adsorption. Microspheres were prepared with a concentration of 5% (w/v) PVA and 0.1% (w/v) PEI 25 000. Images A and C are scanning electron micrographs showing the microsphere surface morphology before (A) and after (C) incubation with plasmid DNA at a target loading level of 1 µg DNA/mg microspheres. Images B and D are z-section confocal microscope images which show the presence of Oregon Green 488 carboxylic acid succinimidyl ester labeled PEI (B) and YOYO-1 iodide-labeled DNA (D) at the microsphere surface. Confocal images were taken after repeatedly washing the microspheres with buffer to remove loosely bound material. Table 2. Effect of PEI Molecular Weight on the Properties of the Microspheres sample

PEI contenta zeta potentialb

control 1% (w/v) LPEI 400 0.21 ( 0.02 1% (w/v) PEI 2000 1.16 ( 0.21 1% (w/v) PEI 25 000 2.44 ( 0.15

-8.5 ( 0.5 -5.2 ( 1.0 2.2 ( 1.2 4.6 ( 0.2

DNA adsorption capacityc 0.17d 0.14 ( 0.06 1.75 ( 0.11 2.57 ( 0.17

a Expressed as µg PEI/mg microspheres. b Expressed in mV, measured in 10 mM phosphate buffer, pH 7.4. c Expressed as µg DNA/mg microsphere (measured in 10 mM phosphate buffer, pH 7.4 at a loading level of 3 µg/mg microspheres). d Average of triplicate samples from one batch.

PVA can also become adsorbed from the aqueous phase on the newly formed microparticle solid interface. The measurements of hydrodynamic layer thickness of PVA adsorption from water to the solid hydrophobic (polystyrene) surface indicate that the layer thickness of the polymer with comparable molecular weight to that used in our study, would be on the order of more than 10 nm.27 It is not possible from the present study to infer what the PVA thickness layer would be. However, the zeta potentials of the control microspheres, prepared with PLG polymer containing free carboxyl endgroups (Table 4), are substantially reduced when compared to surfactant-free PLGA particles from our previous studies,25 indicating formation of a relatively thick PVA layer. Moreover, the observed colloidal stability of the control microparticles, despite the relatively low zeta potential values of about -8.5 mV in low ionic strength buffer (Table 2), further confirm the presence of a PVA layer with a thickness capable of steric stabilization of micrometer-sized particles. Considerably less has been reported in the literature on the adsorption of PEI at solid interfaces in a competitive situation with another polymer. There have been few published reports on using mixtures of polycation and nonionic surfactant solutions, the most similar one to our work being the preparation of PLG nanospheres from a mixed chitosan-PVA aqueous phase.21 Moreover, as discussed above for PVA, the interface at which

the adsorption process occurs changes from a liquid-liquid to a solid-liquid interface during the course of microsphere production. The accumulation of PEI at the liquid-liquid interface can be indicated from the findings that PEI (MW 25 K) can increase the stability of water in oil emulsions, which suggests some activity at the aqueous-organic interface.18 This would indicate that, in the case of PLG microspheres, a competitive adsorption of PVA and PEI may already be occurring at the early stage of their formation when a liquid-liquid interface is present. The HMW PLG used in this study is an end-capped polymer with ester groups; hence, there would be no free terminal carboxyl groups in the initial material that would create surface charge on the formed microspheres. However, exposure of the polymer to an aqueous environment during the production could have initiated hydrolytic degradation and to some extent the creation of free hydroxyl groups. However, the hydrolysis effect would not be expected to be prominent for this higher molecular weight material during a relatively short preparation procedure. The surface potential values for HMW PLG microparticles indicate the presence of surface negative charge (Table 4), but the values are lower than for LMW PLG. Consequently, electrostatic interactions are unlikely to play a major role in the adsorption of PEI on these microspheres, leaving nonelectrostatic interactions to be the probable dominant attractive forces. Recent studies also strongly suggest that there is a significant nonelectrostatic component to PEI adsorption on a range of surfaces.19,20 Effect of PEI MW on Microsphere Properties. To examine the effect of polycation molecular weight on the resultant (18) De Rosa, G.; Quaglia, F.; La Rotonda, M. I.; Appel, M.; Alphandary, H.; Fattal, E. J. Pharm. Sci. 2002, 91(3), 790–799. (19) Meszaros, R.; Thompson, L.; Bos, M.; de Groot, P. Langmuir 2002, 18(16), 6164–6169. (20) Schneider, M.; Brinkmann, M.; Mohwald, H. Macromolecules 2003, 36(25), 9510–9518. (21) Kumar, M.; Bakowsky, U.; Lehr, C. M. Biomaterials 2004, 25(10), 1771– 1777.

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Figure 3. Effect of PEI molecular weight on the CN- ion (negative, m/z 26) and CH2CH2NH2+ (positive, m/z 44) peak intensity. Red, PEI 25 000; blue, PEI 2000; green, LPEI 400; yellow, control. Peak intensity was normalized to total ion count.

microsphere properties, three different PEI polymers were compared: a linear PEI 400 Da, branched PEI 25 000 Da, and smaller branched PEI 2000 Da (Table 2). The trend observed shows that, as the PEI molecular weight increases, there is a consequent increase in the polycation content at the microsphere surface, reflected in higher positive zeta potential. This results in enhanced DNA adsorption capacity for PEI 25 000 Da polymer. However, it must be noted that the increased DNA loading capacity of the microspheres prepared with PEI 25 000 may not solely be attributable to the increased surface PEI content. It may be possible that, with increased size of the polycation molecule, it protrudes perpendicular to the microsphere surfaces further into the DNA solution, which makes it more accessible to interactions with DNA. In addition, the protrusion of PEI decreases possible steric hindrance generated by PVA, with this effect being dependent on the thickness of the PVA layer. It has also been reported that higher molecular weight PEI has greater DNA condensing ability.22 Tof-SIMS analysis confirms that the microsphere polycation content increases with molecular weight. Figure 3 is a plot of the relative intensity of the two principal secondary ions derived from PEI (m/z 44 in the positive spectra, which corresponds to CH2CH2NH2+, and at m/z 26 in the negative spectra, which corresponds to CN-), for the different microsphere delivery systems. Little or no signal was observed for control microspheres at either m/z 26 or m/z 44. However, microspheres surfacemodified with PEI had peaks at both m/z 26 in the negative ion spectra and m/z 44 in the positive ion spectra, which correspond to the two nitrogen-containing secondary ion fragments. Additionally, the relative intensity of the signals increased with increasing chain length of the PEI molecule, confirming that the surface amount of PEI increases with molecular weight, as demonstrated in Table 2. Such data are consistent with theoretical predictions, suggesting that it is entropically more favorable for one large molecule to adsorb when compared to adsorption of multiple small molecules.23 Effect of Particle Size on Microsphere Surface Properties. With potential application of the system designed in this work as a carrier in plasmid DNA vaccination, one should take into account that particle size has been reported to have an affect on (22) Petersen, H.; Kunath, K.; Martin, A. L.; Stolnik, S.; Roberts, C. J.; Davies, M. C.; Kissel, T. Biomacromolecules 2002, 3(5), 926–936. (23) Hesselink, F. T. J. Colloid Interface Sci. 1977, 60(3), 448–466.

Table 3. Effect of Particle Size and PEI Molecular Weight on Microsphere Propertiesa

sample

average size DNA adsorption (µm) PEI contentb zeta potentialc capacityd

control PEI 2000 PEI 25 000 control PEI 2000 PEI 25 000

4.0 4.0 4.0 1.8 1.8 1.8

0 1.16 ( 0.21 2.44 ( 0.15 0 0.45 ( 0.03 1.42 ( 0.07

-8.5 ( 0.5 2.2 ( 1.2 4.6 ( 0.2 -5.6 ( 1.6 0.3 ( 1.1 4.5 ( 0.1

0.17d 1.75 ( 0.11 2.57 ( 0.17 0.17e 1.78 ( 0.09 2.59 ( 0.23

a PEI modified microspheres were prepared with a PEI concentration of 1% (w/v). Results represent the mean ( 1 standard deviation of three batches with three samples per batch. b Expressed as µg PEI/mg microspheres. c Expressed in mV. d Expressed as µg DNA/mg microspheres. e Average of triplicate samples from one batch.

Table 4. Effect of PLG End Group Functionality on the Properties of the Microspheresa sample control (HMW) control (LMW) PEI 2000 (HMW) PEI 2000 (LMW) PEI 25 000 (HMW) PEI 25 000 (LMW)

PEI contentb zeta potentialc 0 0 0.45 ( 0.03 1.52 ( 0.41 1.42 ( 0.07 6.04 ( 0.48

-5.6 ( 1.6 -10.3 ( 0.8 0.3 ( 1.1 0.6 ( 1.5 4.5 ( 0.1 5.6 ( 0.1

DNA adsorption capacityd 0.81e 0.17e 1.82 ( 0.07 2.24 ( 0.22 2.59 ( 0.23 4.63 ( 0.41e

a PEI modified microspheres were prepared with a PEI concentration of 1% (w/v). Results represent the mean ( 1 standard deviation of three batches with three samples per batch. b Expressed as µg PEI/mg microspheres. c Expressed in mV. d Expressed as µg DNA/mg microspheres. Average of triplicate samples from one batch. e DNA loading level of 5 µg DNA/mg microsphere used as loading 100% efficient at the 3 µg/mg load used for the other experiments.

the immunogenicity of the formulations.5 Therefore, microspheres with average sizes of 1.8 and 4.0 µm, those within the range appropriate for particle uptake by antigen-presenting cells, were compared (Table 3). The data indicate that, as particle size is reduced, the PEI content per mass of the microspheres decreases. This is despite the fact that surface area values of the microspheres are more than 4-fold higher for values for the smaller microspheres. These data are in agreement with a study on polyelectrolyte adsorption that predicts that the adsorption of polyelectrolyte increases as particle size increases.24 This phenomenon is attributed to the charged polyelectrolyte chains being able to

Surface Modification of Microspheres

flatten and become more extended along the surface of bigger particles, thus losing less chain entropy on adsorption. Interestingly, the reduction in surface PEI content for 1.8 µm microspheres did not significantly reduce the DNA loading capacity, expressed per mass of microspheres. This may be due to increased microsphere surface area (per mass) which compensates for the reduced PEI surface content, resulting in overall DNA loading being largely unaffected. Effect of PLG Carboxyl Endgroup. Further investigation was undertaken to assess the potential effect that the nature of the PLG polymer, i.e., the presence of the carboxyl end group, has on the microsphere surface properties, primarily the PEI surface accumulation. The microspheres produced from an endcapped high molecular weight polymer (HMW PLG, 30-70 000 Da) were therefore compared to those produced from free carboxyl endgroups containing low molecular weight polymer (LMW PLG, 16 500 Da). The aim was to produce microspheres with identical sizes; however, in practice, although the overall distribution profile was similar, the microspheres prepared from the LMW PLG were slightly smaller: 1.5 µm compared with 1.8 µm. The data summarized in Table 4. illustrate that microspheres from the LMW PLG have a greatly increased PEI content compared to those produced from the HMW PLG. The enhanced PEI content was associated with an increased positive zeta potential and an increased DNA loading ability, regardless of polycation MW. It is likely that the greater PEI content of the LMW PLG microspheres can be attributed to the presence of the free terminal carboxyl groups. At least some of these groups would be present at the microsphere surface, 25 and their presence accounts for the higher negative zeta potential values of the control microspheres prepared with LMW PLG, when compared to the HMW PLG (Table 4). Electrostatic interactions with ionized carboxyl endgroups in aqueous medium would provide a driving force for adsorption of PEI. Further to the discussion above, it can be suggested that some PEI would be present at the “future” microparticle surfaces from its accumulation at the liquid-liquid interface in the emulsion stage of the production, and some could be adsorbed because of electrostatic interactions with the charged groups present once the surface is formed. Electrostatic interaction with the surface charges would neutralize PEI on its adsorption and hence reduce intermolecular repulsion between neighboring PEI molecules, resulting in the higher amount adsorbed. In that context, a more than a 3-fold increase in the PEI content for LMW microspheres did not, however, result in a proportional increase in the DNA association at the surface. This may be because a number of PEI charges will be engaged in electrostatic interactions with surface carboxyl groups and thus are not available for DNA binding. Colloidal Stability on DNA Adsorption. The microspheres designed in this work are intended to be a generic delivery system capable of loading different nucleic acid structures, DNA or RNA, following simple incubation with the microsphere suspension. However, the colloidal stability of the final product needs to be maintained for the suspension to be injectable and loaded microspheres taken up by the cells. Binding of DNA to the surface of cation-modified microspheres would reduce their positive charge and hence compromise their colloidal stability, assuming it is primarily maintained by electrostatic repulsion. In the present work, the maintenance of colloidal stability relies on the presence of PVA at the microspheres’ surfaces to provide steric stabilization to the system. Furthermore, adsorption of polyanions to positively charged particles causes aggregation, as it may be entropically (24) Chodanowski, P.; Stoll, S. J. Chem. Phys. 2001, 115(10), 4951–4960. (25) Stolnik, S; Garnett, M. C.; Davies, M. C.; Illum, L.; Bousta, M.; Vert, M.; Davis, S. S. Colloids Surf., A: Physicochem. Eng. Aspects 1995, 97, 235–245.

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favorable for polyanions to bind to two different particles rather than imposing the conformational restrictions necessary to bind to just one particle.26 Aggregation is likely to be favored when particles are able to come into close contact with one another. In the present study, the surface presence of PVA should provide a situation where chains on two approaching microparticles resist overlap and create a repulsive force to separate them to a distance where the attractive interaction would be too weak to keep the particles together. Figure 4 illustrates the colloidal stability studies. It should be noted that there was no aggregation observed for control microspheres (no PEI) after incubation with DNA. Images show that, in the case of PEI 2000, while no agglomeration occurred following DNA adsorption to the larger particles prepared with HMW PLG, some was noted following DNA addition to the smaller HMW PLG microspheres, and marked aggregation was noted for LMW PLG particles. Similar results were obtained for microspheres surface-modified with PEI 25 000; however, the agglomeration was greater for the smaller microspheres prepared from both PLG polymers. In summary, as the particle size is reduced and molecular weight of the PEI increases, the steric effect provided by the PVA layer is no longer able to prevent aggregation. The destabilizing effect of particle size reduction can be, at least in part, a consequence of the increased number (in the same mass of microparticles and suspending volume) and motility of the smaller particles. Also, conformational restrictions imposed on adsorbing DNA molecules may be greater on the smaller particles, which may cause DNA bridging between microspheres to become more favorable. A similar phenomenon was reported for DNAinduced aggregation of microspheres to which PEI was covalently attached.10 It should be noted that, in that study, the aggregation could only be prevented when particles of approximately 7 µm were used, which are potentially too big for uptake by antigenpresenting dendritic cells,33 while in the present study, 4.0 µm microparticles remained colloidally stable. Similar beneficial effects of steric stabilization were reported for cationic polystyrene latex which aggregated upon addition of an oligonucleotide, but in this case, the coating with nonionic surfactant (poloxamer 338) prior to the polyanion addition greatly enhanced the colloidal stability.28 Similarly, PEI-modified PLG nanospheres prepared in the presence of Tween 80 and Poloxamer 188 were found to be colloidally stable on plasmid DNA adsorption,13 while PLA and poly(L-lysine)-graft-dextran nanoparticles were found to have enhanced colloidal stability when compared to particles prepared with the poly(L-lysine) homopolymer alone.11 The decreased colloidal stability observed when the molecular weight of the PEI molecule was increased is likely to be attributed to a combination of the increased concentration and different conformation of the polycation at the microsphere surface. PEI 25 000 may adopt a more extended conformation than PEI 2000, which may reduce the steric effect provided by the PVA and thus, on addition of DNA allow particles to come into close enough contact for aggregation to occur. In an attempt to reduce agglomeration of PEI 25 000 surface-modified microspheres following DNA addition, the PVA concentration and molecular weight were increased (data not shown); however, neither of the parameters enhanced colloidal stability of the microspheres on DNA addition. (26) Lyklema, J.; Fleer, G. J. Colloids Surf. 1987, 25(2-4), 357–368. (27) Fleer,. G. J.; Cohen Stuart, A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman and Hall: London, UK, 1993. (28) Gotting, N.; Fritz, H.; Maier, M.; von Stamm, J.; Schoofs, T.; Bayer, E. Colloid Polym. Sci. 1999, 277(2-3), 145–152.

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Figure 4. PLG microsphere aggregation following DNA adsorption (3 µg DNA/mg microspheres). A-C, control microspheres; D-F, 1% (w/v) PEI 2000 surface-modified microspheres; G-I, 1% (w/v) PEI 25 000 surface-modified microspheres. (A,D,G) HMW PLG, 4 µm. (B,E,H) HMW PLG, 1.8 µm. (C,F,I) LMW PLG, 1.8 µm.

Effect of Buffer on DNA Adsorption. For the microspheres designed in this study to be administered parenterally, the suspension medium needs to be of appropriate osmolarity. Hence, in a separate study, the effect of the suspension buffer on DNA adsorption was investigated (Table 5), particularly as the driving force for DNA loading is believed to be electrostatic in nature. The results indicate that there are no substantial differences in the loading capacity of the microspheres in the buffers acceptable for the parenteral administration, whereby in relatively high ionic strength buffers the DNA loading was not compromised relative to 10 mM phosphate buffer. This implies that nonelectrostatic forces may be involved in DNA adsorption onto PEI-modified microspheres, as charge screening would reduce adsorption if electrostatic forces were solely responsible for the attraction.23 Furthermore, a high concentration of counterions in the NaCl saline buffer may increase the flexibility of the DNA molecule by screening the intramolecular repulsion. Consequently, DNA may behave more like an uncharged polymer, allowing for increased loop and tail formation and closer packing of the DNA at the microsphere surface.23,26 Effect of DNA Concentration on Loading. Table 6 shows the effect of initial DNA concentration on the microsphere (4 µm) DNA loading capacity in 0.9% (w/v) sodium chloride. The data illustrates that, as the initial DNA concentration increases from 3 to 10 µg DNA/mg of microspheres, higher DNA loading

can be achieved. However, the balance between the loading and “incorporation efficiency” (proportion of initially present DNA associated with the final formulation) is essential for a welldesigned delivery system. The microspheres prepared with PEI 25 000 Da show almost 100% loading efficiency at a target DNA loading of 3 or 5 µg DNA/mg microspheres. At the 10 µg/mg target load, although incorporation efficiency was lower, a DNA binding capacity of >8 µg DNA/mg particles was achieved. In itself, this is an encouraging compromise that achieves a very high loading with acceptable losses of DNA. However, one should note that, as the amount of surface-loaded DNA increases, the zeta potential of the final microspheres becomes more negative. This phenomenon could potentially contribute to the colloidal stability of the formulation, as reported previously;28 however, it is indicative of the presence of DNA that is not well-complexed at the microspheres’ surfaces. Insufficient DNA condensation may potentially have a negative effect on the protection of the DNA against enzymatic degradation following parenteral administration and consequently its therapeutic effect, as we reported earlier.29 Furthermore, the comparison of zeta potentials for PEI 2000 and PEI 25 000 modified microspheres indicates that DNA is complexed to a greater extent on the latter at similar loading (29) Chim, Y. T. A.; Lam, J. K. W.; Ma, Y.; Armes, S. P.; Lewis, A. L.; Roberts, C. J.; Stolnik, S.; Tendler, S. J. B.; Davies, M. C. Langmuir 2005, 21, 3591–3598.

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Figure 5. Phagacytosis of PEI 25 000 surface-modified microspheres before and after GFP plasmid DNA loading. Pictures show phase contrast (A,C,D) and fluorescent (B,E,F) images of the uptake of PEI 25 000 surface-modified microspheres by MonoMac 6 cells before (A-C) and after GFP plasmid DNA adsorption. In images A-C, microspheres were labeled with coumarin 6, and in images D-F, GFP plasmid DNA was labeled with YOYO-1 iodide. Images C and F are x-y scans which show the presence microspheres inside the MonoMac 6 cells. Table 5. Effect of Buffer System on GFP DNA Loading Ability of HMW PLG Microspheres (1.8 µm) Surface-Modified with 1% (w/v) PEI 25 000a diluent

loading (µg DNA/mg microspheres)

phosphate buffer, (10 mM PB) phosphate buffered saline (PBS) saline [0.9% (w/v) NaCl] sucrose [10% (w/v)]

1.74 ((0.01) 2.08 ((0.09) 2.48 ((0.04) 1.82 ((0.13)

a

Results represent the mean ( 1 standard deviation of triplicate samples.

levels, hence presenting this system as a better option for further in ViVo studies. It is important to note that some adsorption of DNA to control microspheres (no PEI) occurs when saline was used as the medium. This resulted in highly negative zeta potential values as there are no opposing positive charges to neutralize the DNA. Microspheres and DNA Entry into Cells in Culture. The images in Figure 5 clearly demonstrate that the PEI 25 000 modified microspheres loaded with GFP are able to enter, and more importantly, deliver loaded DNA into cells in culture. The images illustrate that DNA is associated with the microspheres within the cells at the early time point following the uptake and thus the microspheres are responsible for the delivery of DNA. The reasoning behind the use of GFP plasmid in the present study was to eventually assess the level of transfection following the application of the microspheres to the phagocytic cells in culture. However, the level of reported gene expression was not measurable. The MonoMac 6 cells, used in the present study as model phagocytic cells, have previously been reported to be “refractory” to transfection by calcium phosphate or DEAE dextran, hence making the reporter gene assays difficult to

Table 6. Summary of the Effect of GFP Plasmid DNA Adsorption on the Properties of PEI Modified Microsphere (4 mm) Formulationsa sample control

PEI 2000

PEI 25 000c

DNA concentrationb no DNA 3 5 10 no DNA 3 5 10 no DNA 3 5 10

DNA loadingb 0.68 1.05 1.75 2.29 ( 0.48 3.25 ( 0.37 3.80 ( 0.18 2.98 ( 0.02 4.98 ( 0.01 8.19 ( 0.19

zeta potential size (µm) (mV) mean median -4.8 ( 0.9 4.18 -47.5 ( 1.5 4.16 -45.8 ( 3.2 4.17 -42.6 ( 3.3 3.84 0.6 ( 0.5 3.55 -29.9 ( 5.6 9.04 -35.0 ( 0.8 5.09 -25.6 ( 6.1 6.09 5.3 ( 0.8 5.61 -0.6 ( 0.2 7.82 -8.2 ( 1.2 11.27 -29.3 ( 0.7 7.71

4.23 4.23 4.21 3.91 3.58 8.06 3.93 4.60 4.58 6.27 9.23 5.24

a Results represent the mean ( 1 standard deviation of three samples. Expressed as µg DNA/mg microspheres. c Microspheres were prepared with a PEI concentration of 1% (w/v). b

perform.30 Similarly to our case, no measurable transfection was observed for cationic PLG microparticles applied to primary macrophages in cell culture, despite the particle uptake by the cells, and only a minor number of cells were transfected in a murine macrophage cell line.31 Furthermore, following transfection of bone marrow-derived dendritic cells with a reporter gene surface adsorbed onto the surface of the microspheres modified with cationic surfactant, CTAB, there was no detectable level of reporter gene activity. However, in this case, further studies confirmed the gene expression by RT-PCR analysis and (30) Klan, N; Steinhilber, D. Biotechniques 2003, 34, 142–7. (31) Walter, E; Merkle, H. P. J. Drug Targeting 2002, 10, 11–21.

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antigen presentation by the transfected cells.32 Hence, considering current literature on transfection of antigen presenting cells, our results agree in demonstrating the microsphere uptake and cellular localization of the adsorbed DNA. However, further antigen presentation studies in Vitro and in ViVo assessment of immune response in animal models, would provide an ultimate assessment of the potentials of our current system.

Conclusion Microspheres were prepared in this work by a classical o/w solvent evaporation method where the microspheres surfaces were successfully modified to simultaneously introduce polycationic and steric stabilizing components by simple addition of (32) Denis-Mize, K. S.; Dupuis, M.; MacKichan, M. L.; Singh, M.; Doe, B.; O’Hagan, D.; Ulmer, J. B.; Donnelly, J. J.; McDonald, D. M.; Ott, G. Gene Ther. 2000, 7, 2105–2112. (33) Foged, C; Brodin, B.; Frokjaer, S.; Sundblad, A. Int. J. Pharm. 2005, 298, 315–322.

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these materials into the water phase. A range of polycations can be used to achieve subsequent surface loading of DNA, where PEI was used as a model polycation in the further systematic studies. It was shown that, as the preparation concentration of the polycation increases from 0.1 to 0.5 (w/v) and its molecular weight increases from 400 to 25 000 Da, the final microsphere zeta potential and DNA loading ability both increase. Decreasing the particle size reduced the microsphere PEI content and increased the tendency for particles to agglomerate on DNA addition. Optimization of design parameters: steric stabilization, particle size, and surface DNA loading demonstrated the feasibility using an established o/w method of producing cationic microspheres capable of delivering DNA into the cells for the purpose of DNA vaccination. Acknowledgment. The authors would like acknowledge BBSRC and AstraZeneca for joint funding. LA703735N