hydrophilic poly(dl-lactide-co-glycolide) microspheres for the delivery of dna to human-derived...

Journal of Controlled Release 76 (2001) 149–168www.elsevier.com/ locate / jconrel

Hydrophilic poly(DL-lactide-co-glycolide) microspheres for thedelivery of DNA to human-derived macrophages and dendritic

cellsa , b c a*Elke Walter , Donatus Dreher , Menno Kok , Lars Thiele ,

d d aStephen Gitahi Kiama , Peter Gehr , Hans Peter MerkleaDepartment of Applied Biosciences, Swiss Federal Institute of Technology Zurich (ETH), Winterthurerstrasse 190, 8057 Zurich,

SwitzerlandbDepartment of Genetics and Microbiology, University of Geneva, 1 Rue Michel-Servet, 1211 Geneva 4, SwitzerlandcDivision of Pneumology, University Hospital of Geneva, 24 Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland

d ¨Institute of Anatomy, University of Berne, Buhlstrasse 26, 3000 Berne 9, Switzerland

Received 15 December 2000; accepted 13 June 2001

Abstract

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres have a proven track record for drug delivery and aresuggested to be ideal carrier systems to target therapeutics into phagocytic cells such as macrophages (MFs) and dendriticcells (DCs). Microspheres prepared by spray-drying from different PLGA-type polymers were evaluated regarding theireffect on phagocytosis, intracellular degradation and viability of human-derived macrophages MFs and DCs. Even themicrospheres prepared from the most hydrophilic polymer RG502H, were efficiently phagocytosed by primary human MFsand DCs. Interestingly, uptake of PLGA microspheres by DCs as potent immune modulator cells was almost as efficient asuptake by the highly phagocytic MFs. Phagocytosed microspheres remained inside the cells until decay with none of themicrosphere preparations induced significant apoptosis or necrotic cell death. Acidic pH and the phagosomal environmentinside the cells enhanced microsphere decay and release of encapsulated material. Degradation of microspheres consisting ofthe most hydrophilic PLGA polymer RG502H occurred in a reasonable time frame of less than 2 weeks ensuring the releaseof encapsulated drug during the life span of the cells. To explore important technical and biological aspects of DNAmicroencapsulation, we have studied DNA loading and in vitro DNA release of microspheres from different PLGA typepolymers. Hydrophobicity and molecular weight of the PLGA polymers had profound influence on both the encapsulationefficiency of DNA and its release kinetics in vitro: the hydrophilic polymers showed higher encapsulation efficiency andfaster release of intact DNA compared to the hydrophobic ones. These results suggest that microspheres from the PLGApolymer RG502H have improved characteristics for DNA delivery to human MFs and DCs. 2001 Elsevier Science B.V.All rights reserved.

Keywords: Dendritic cells; DNA; Macrophages; Poly(lactide-co-glycolide) (PLGA) microspheres; Toxicity

*Corresponding author. Tel.: 141-1-6356-013; fax: 141-1-6356-881.E-mail address: [email protected] (E. Walter).

0168-3659/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 01 )00413-8

150 E. Walter et al. / Journal of Controlled Release 76 (2001) 149 –168

1. Introduction A very promising application of PLGA would bethe delivery of DNA for genetic vaccination [20,21].

Biodegradable polymers based on poly(lactide-co- Recently, different approaches have been reportedglycolide) (PLGA) are fully biocompatible and there- for the incorporation of DNA into PLGA-typefore among the most commonly used materials for microspheres [9,17,18,22–25]. In a previous study,the microencapsulation of therapeutics [1,2]. PLGA we explored microencapsulation of DNA by spray-microspheres have been used for antigen delivery drying using a PLGA (50:50) polymer [17]. Weand for the stimulation of immune responses [3–5]. obtained small size microspheres with most of theSmall size microspheres are efficiently phagocytosed DNA concentrated inside of the particle core and to aby macrophages (MFs) and are capable of inducing minor extent located at the particle surface. Mi-a cytotoxic T cell response in vitro and in vivo croencapsulated plasmid DNA extracted from micro-[5–7]. Furthermore, the feasibility of biodegradable spheres and released during the burst release phasemicrospheres for DNA delivery has been confirmed was able to transfect cells in vitro. In this work, weunder in vivo conditions [8–10], but detailed in- studied the influence of different PLGA type poly-formation about the interaction of the delivery mers on microsphere uptake and degradation bysystem and target cells is still lacking. human MFs and DCs, derived from peripheral blood

Dendritic cells (DCs) have been demonstrated to monocytes. The use of different polymers for theplay a central role in controlling immunity as preparation of DNA-containing microspheres byprofessional antigen presenting cells (APCs) [11]. spray-drying was evaluated.They display 10 to 100 times more major histocom-patibility complex (MHC) molecules on their surfacethan other APCs and offer a powerful tool to 2. Materials and methodsmanipulate the immune system [12]. DCs can inter-nalise soluble antigens, whole microbes, apoptotic 2.1. Materialsbodies, and to a certain extent, synthetic latexmicroparticles [13–15] but it is still unclear whether Poly(D,L-lactide-co-glycolide) type polymersbiodegradable microspheres are efficiently engulfed (PLGA, Resomer RG502H, RG502, RG503H,by DCs and how these would be processed inside the RG503, RG752, R202, R202H) were purchased fromcells. A recent report demonstrated that inflammatory Boehringer Ingelheim (Ingelheim, Germany). Crudemonocytes phagocytose latex particles and differen- salmon testes DNA was obtained from Sigmatiate into DC type cells while migrating to the (Buchs, Switzerland). PicoGreen quantification assaydraining lymph nodes [16]. These findings stress the for double-stranded DNA (dsDNA) was obtainedimportance of investigating biodegradable micro- from Molecular Probes (Lucerne, Switzerland).spheres as a delivery system to target DCs. Green fluorescent protein (GFP) reporter gene plas-

The type and the molecular weight of the PLGA mid was generated by cloning the GFP gene into thepolymers and the encapsulation technology largely VR1012 vector. Plasmid DNA was prepared withinfluence the encapsulation efficiency and the release Qiagen (Basel, Switzerland) endotoxin-free buffersof encapsulated material from microspheres according to the manufacturer’s instructions and[4,17,18]. Generally, the release of antigen from resuspended in water. Materials for cell culturePLGA microspheres correlates well with the degra- experiments were obtained from Life Technologiesdation of the polymer and can be sustained up to 20 (Basel, Switzerland). All other chemicals used wereweeks. Microspheres prepared from more hydro- of analytical grade (from Fluka, Buchs, Switzerland)phobic polymers are degraded slower and due to unless otherwise specified.their hydrophobic surface are suggested to be moresusceptible for phagocytosis by MFs [4,19]. On the 2.2. Preparation of microspheresother hand, when targeted to APCs, the limited lifespan of the cells requires the delivery of the encapsu- PLGA microspheres were prepared by spray-dry-lated material in a reasonable time frame of up to 1 ing as described in detail elsewhere [17]. Briefly,month. DNA was dissolved in water or in 0.1 M NaHCO3

E. Walter et al. / Journal of Controlled Release 76 (2001) 149 –168 151

and the resulting solution was dispersed in 2.5, 5 or (Mastersizer X, Malvern Instruments) and calcula-10% (w/w) polymer solution in ethyl formate (EF) tion was based on Mie’s theory accounting for theor methylene chloride (MC) by means of an ul- optical properties of the polymer.

trasonic processor (Vibra-Cell , Sonics & Materials,Danbury, CT, USA) forming a water in oil (W/O) 2.4. Cell culturedispersion. Plain microspheres were prepared with-out adding DNA to the aqueous phase using MC as a DCs and MFs were obtained from humansolvent. W/O dispersions were spray-dried in a peripheral blood monocytes (PBMCs) according to

¨laboratory spray dryer (Model 190, Buchi, Flawil, Sallusto et al. [28]. Briefly, PBMCs were isolatedSwitzerland). The microspheres were washed and from buffy coats prepared from healthy donorsdried under vacuum for 24 h. For pH measurements (Blood Bank Zurich, Switzerland) by density gra-the aqueous phase (containing 1 mg/ml salmon dient centrifugation on Ficoll-Paque (research grade,DNA) of the W/O dispersion was separated by Pharmacia Biotech). PBMC were resuspended incentrifugation directly after ultrasonication and the RPMI 1640 medium supplemented with 10%pH value was determined by using a pH electrode (pooled) human serum (Blood Bank Zurich) and(pH meter 240, Corning). then allowed to adhere for 2 h in cell culture flasks.

For morphological examination, the microspheres Non-adherent cells were removed and adherent cellswere mounted on double-faced adhesive tape, sput- were cultured in RPMI 1640 supplemented with 5%tered with platinum and viewed in a Hitachi S-700 human serum, 2 mM glutamine, 50 IU/ml penicillinscanning electron microscope. and 50 mg/ml streptomycin. DCs were generated by

Fluorescent-labeled microspheres were prepared culture in the presence of 1000 IU/ml IL-4 (Sigma)by adding 6-coumarin (5 mg/g polymer, Acros and 50 ng/ml GM-CSF (R1D Systems), whereasOrganics, Geel, Belgium) to the organic phase. MFs were obtained without any additional supple-Leakage of 6-coumarin from microspheres was ments. Cultures were kept at 378C in 5% CO2

verified as described before [26] and was found to be humidified atmosphere overnight and were mechani-5inferior to 1%. cally removed and reseeded onto 24-well plates (10

cells /well) for further studies. In all experiments2.3. Rose Bengal partitioning method [27] involving flow cytometry, DCs were cultured in

heat-inactivated fetal calf serum (FCS, 10%), asDifferent concentrations of microspheres (2–10 described previously [28], instead of the human

mg/ml) were suspended in a solution of 10 mg/ml serum. Immature and lipopolysaccharide (LPS)-Rose Bengal in 0.1 M phosphate buffer, pH 7.4. stimulated DCs were characterized by fluorescence-Samples were incubated at room temperature for 3 h, activated cell sorter (FACS) analysis of surfacecentrifuged at 14 000 rpm and the amount of free expression of CD11b, CD80, CD86, and CD40Rose Bengal in the supernatant was determined according to Cochand et al. [29]. According to thesespectrophotometrically at 542.7 nm. The partition criteria, more than 90% of the cells were identifiedquotient (PQ) was calculated by dividing the amount as DCs.of Rose Bengal bound on particle surface by theamount of Rose Bengal in dispersion medium. 2.5. Phagocytosis studiesPlotting of PQ against the total surface area of themicrospheres yields a straight line. The slope of this Cells were used 8–10 days after seeding forline is used as an indicator of the hydrophobicity of phagocytosis studies. Fluorescent-labeled micro-the particle surface [27]. Slopes were calculated by spheres were suspended in cell culture medium and

5linear regression analysis. The total surface area of added to the cells at a concentration of 5310the particles was calculated from the particle size particles per well in a 24-well plate. Polystyrenedistribution after assessing particle density by using a beads (Polyscience, Warrington, PA, USA) with agas displacement pycnometer (AccuPyc 1330, Mi- mean diameter of 4.5 mm were used as controls.cromeritics, Belgium). The particle size distribution Incubation was performed either at 37 or at 48C forwas determined by laser light scattering different time periods. Cells were washed with RPMI


1640 and fixed with 3% paraformaldehyde in phos- translocated to the outer layer of the cell membranephate-buffered saline (PBS) prior to microscopic in the early stages of apoptosis [30]. Necrosis wasanalysis. Data indicate the mean (S.E.) number of detected by propidium iodide (PI, 1 mg/ml, PharM-particles counted in 150 cells. ingen, San Diego, CA, USA), which penetrates

For intracellular degradation studies, microscopic necrotic cells and stains their DNA. Cells wereexamination was directly performed in the 24-well washed and exposed during 10 min at room tempera-plates without any further treatment of the cells. The ture to annexin V-PE in annexin-binding buffer (10inverse microscope (Axiovert 35, Zeiss) was mg/ml, PharMingen), resuspended in the same bufferequipped for fluorescence microscopy (filter: excita- without annexin V at 48C, and exposed to PI (1tion 450–490 nm/emission 520 nm) and pictures mg/ml, PharMingen) at 48C immediately before thewere taken using a digital camera (Kappa CF8/ analysis. The three-color flow cytometry detecting1DCX, Videal). 6-coumarin, PE, and PI was performed on a FAC-

Scan (Becton Dickinson, Mountain View, CA, USA).2.6. Processing of cells for transmission electron For all experiments, gating was done such as tomicroscopy include all cells of typical DC size and complexity.

Cut-offs for apoptosis and necrosis were derivedCells were washed three times in Hank’s balanced from control cells and maintained throughout the

salt solution and resuspended in 2.2% phosphate- experiment. The sensitivity of annexin-PE and PI tobuffered glutaraldehyde solution at pH 6.8. The cells detect apoptosis and necrosis, respectively, waswere postfixed in 1% osmium tetroxide in 0.1 M checked with C2-ceramide (Sigma) and was shownsodium cacodylate buffer and contrasted in 0.5% to be near 100% [31].uranyl acetate in 0.05 M maleate buffer. Dehydrationwas performed in a graded series of ethanol (70, 80, 2.8. Analysis of DNA96, 100, and 100%) and gradual replacement ofethanol with propylene oxide was carried out before The DNA content was determined with 10–20 mginfiltration and embedding in epoxy resin. Semithin of microspheres dissolved in 1 ml of dimethylsulfox-and ultrathin sections were cut using an ultramicro- ide. The solutions were measured photometrically attome (Reichert, Austria) and were collected on 200 a wavelength of 260 nm. PicoGreen analysis formesh carbon coated copper grids. After staining with dsDNA was performed after dilution with PBS inuranyl acetate and counterstaining with lead citrate, 96-well plates according to the manufacturer’s in-samples were observed with a Phillips 300 transmis- structions, using an automated plate reader (Fluoro-sion electron microscope under an accelerating volt- Count, Canberra Packard). Integrity of encapsulatedage of 60 kV. Semithin sections were collected on plasmid DNA was checked after extraction of theglass slides, stained with 0.5% toluidine blue and DNA as described in detail elsewhere [10]. Theviewed by light microscopy. topological form of plasmid DNA extracted and

released from microspheres was analyzed by 0.6%2.7. Viability assays agarose gel electrophoresis. Gels were electrophor-

esed for 90 min at 80 V/cm in a Tris–acetate–EDTAViability of the cells after phagocytosis of PLGA buffer system (pH 8.0) and DNA was visualized

microspheres was assessed by three-color flow cy- using ethidium bromide staining.tometry, detecting simultaneously the uptake ofmicrospheres, apoptosis, and necrosis. To perform 2.9. In vitro release of DNAthree-color flow cytometry of DCs, microsphereswere loaded with a small amount of 6-coumarin (50 DNA release was determined by suspending 10–mg/g). At different time points after phagocytosis of 20 mg of microspheres in 4 ml of PBS, pH 7.4, pHmicrospheres, cells were labeled with annexin V- 6.2 or pH 5.4 preserved with 0.02% sodium azide.phycoerythrin (PE), to detect apoptosis. Annexin V The particles were wetted prior to suspension usingspecifically binds to phosphatidyl serine, which is polysorbate 20 resulting in a final concentration of


0.25% surfactant in PBS. The vials were fixed with the spray-drying technique, the majority of thehorizontally in a shaker at 378C, and samples were microspheres being below 10 mm in diameter [17].withdrawn at regular time intervals after centrifuga- The surface hydrophobicity of plain PLGA micro-tion and replaced by fresh medium. For reasons of spheres from different types of PLGA polymerclarity, error bars were not plotted in Figs. 7C and (Table 1) was investigated by using the Rose Bengal

¨8B. Standard deviations range between 5 and 15%. partitioning method according to Muller [27]. In thismethod, microspheres suspended in an aqueous

2.10. Transfectivity of plasmid DNA medium are considered as a two-phase system. Thesurface of the particles is one phase and the disper-

Transfection experiments in 293 cells (ATCC sion medium is the second phase. Plotting theCRL-1573) were performed to check the biological partition quotients of the dye against the total surfacefunctionality of DNA plasmid. Cells were main- area of the microspheres yields a straight line, thetained in Dulbecco’s modified Eagle medium with slope of which represents an arbitrary hydrophobicityhigh glucose (4.5 g / l) containing 10% fetal bovine value for the surface of the microsphere (Fig. 1).

serum. DNA was complexed with Superfect Rose Bengal adsorption to the particle surface in-(Qiagen) according to the manufacturer’s instruc- creased with increasing amount of lactic acid in thetions. 24 h after transfection, positive cells were polymer indicating a more hydrophobic surface (Fig.detected by fluorescence microscopy (Axiovert 35, 1). Furthermore, microsphere surfaces from uncap-Zeiss; filter set 450–490/520 nm, beamsplitter 510 ped polymer were significantly more hydrophilicnm) and positive cells were counted out of 10 000– compared to capped polymer (Fig. 1).15 000 cells.

3.2. Phagocytosis and degradation of microspheresin APCs

3. ResultsSurface characteristics of particles greatly influ-

3.1. Surface hydrophobicity of microspheres ence the phagocytosis by APCs, low phagocytosisrates are suggested to correlate with hydrophilic

A wide range of PLGA type polymers has been particle surfaces [32,33]. To investigate whether thecommonly used for the encapsulation of therapeutics. type of polymer influences the uptake by APCs, weThe composition of these polymers not only de- performed phagocytosis studies in human-derivedtermines the release pattern of encapsulated material, DCs and MFs. Efficient phagocytosis of all prepara-but is suspected to also considerably affect the tions of microspheres tested was found with both cellhydrophobicity of the resulting microspheres and types (Fig. 2A and C). No particle uptake wastherefore, may influence the uptake of PLGA micro- obtained when the experiment was performed at 48Cspheres by APCs. Spherical microspheres are formed (Fig. 2B). Transmission electron microscopy studies

Table 1Different PLGA type polymers used for the microencapsulation of DNA

a bPolymer Ratio lactide:glycolide Molecular weight Viscosity

RG502H 50:50 (uncapped, COOH) 14 000 0.17RG502 50:50 (capped, ester) 14 000 0.18RG503H 50:50 (uncapped, COOH) 35 000 0.4RG503 50:50 (capped, ester) 35 000 0.36RG752 75:25 (capped, ester) 17 000 0.22R202H 100 (uncapped, COOH) 14 000 0.2R202 100 (capped, ester) 14 000 0.2

a M according to the supplier.wb In 0.1% chloroform 258C (dl /g) according to the supplier.


4). Microspheres composed of RG502H were de-graded rapidly with significant loss of shape startingat day 4 (Fig. 4A). After 9 days, microspheresdisappeared and enclosed fluorescence marker wasdistributed evenly over the cell volume (Fig. 4A).Phase contrast microscopy confirmed the lack ofparticles inside the cells at day 13 as illustrated inFig. 4. In contrast, microspheres from more hydro-phobic polymers were still clearly detectable asintact particles after 13 days with the fluorescencemarker merely starting to be released and distributedacross the cell for RG502 (Fig. 4). RG752 and R202microspheres remained intact inside the cells (Fig.4). The same results were obtained with DCs.

3.3. Viability of cells after phagocytosis ofmicrospheres

The effect of uptake of different microspherespreparations on the viability of DC was assessed at24 h and at 7 days after the uptake (Table 2). Thefluorescent dye 6-coumarin was used to stain thePLGA (RG502H) microspheres, whereas annexinV-PE and PI detected apoptotic and necrotic cells,respectively (see Materials and methods). Incorpora-tion of the fluorescent dye into the microspheres hadno influence on the percentage of apoptotic ornecrotic cells (data not shown). Even when we useda low concentration of microspheres (10 mg/well),

Fig. 1. Rose Bengal partitioning method with (A) plain and (B)the majority of cells stained positive for 6-coumarinDNA loaded microspheres (nominal loading was 2%). Plot of the(not shown), the uptake by the DCs apparentlypartition quotient versus the total surface area of the microspheres.increasing proportionally to the particle concentra-The slope of the straight line is a measure of the surface

hydrophobicity of the microspheres. Calculated slopes are in- tion (Table 2). However, the microspheres did notcluded as numbers in the graph. increase the proportion of apoptotic or necrotic cells

up to 7 days after phagocytosis (Table 2). At thisclearly confirmed that particles are located inside the point, the phagocytosed RG502H microspheres werecells surrounded by a presumably phagosomal mem- already partly degraded (see Fig. 4A) and the fluores-brane (Fig. 3). cent signal of 6-coumarin had already substantially

A slightly higher particle uptake was observed in decreased, probably due to degradation of the dyeMFs as compared to DCs, when low amounts of (Table 2).particles were applied to the cells (Fig. 2C). Interest-ingly, microspheres composed of the most hydro- 3.4. Influence of polymer type on thephilic polymer RG502H were phagocytosed as effi- encapsulation of DNAciently as more hydrophobic microspheres in DCs(Fig. 2C). The encapsulation of DNA into PLGA micro-

The intracellular degradation of microspheres in- spheres as a potential delivery system for DNAside MFs and DCs was investigated in cell culture vaccines has raised increasing interest in the recentexperiments over a time span of up to 13 days (Fig. past [9,10,34]. Therefore, we selected different


Fig. 2. Phagocytosis of microspheres by human-derived MFs and DCs. (A) Uptake of fluorescent-labeled PLGA (RG502H) microspheres(100 mg/24-well) after 4 h of incubation at 378C. Cells were examined simultaneously by phase contrast (left panel) and fluorescence (rightpanel). (B) Control experiments were performed at 48C showing no particle uptake by MFs (see figure) and DCs (not shown). (C) Forquantification of particle uptake, phagocytosis studies were performed at 378C as described in Materials and methods. Bar520 mm.

PLGA type polymers varying in the composition and solvent and DNA/water phase is generated whichmolecular weight (Table 1) for the preparation of was subsequently spray-dried to form the micro-DNA-containing microspheres by spray-drying. In a spheres. Two commonly used solvents for spray-previous study, we demonstrated comparable per- drying, ethyl formate and methylene chloride, wereformance of circular plasmid DNA and linear salmon employed. In a recent study, we demonstrated thatDNA encapsulated in microspheres [17]. Further- DNA was irreversibly inactivated at low pH [17]. Asmore, salmon DNA was shown to be more suscep- hydrophilic uncapped PLGA type polymers (Tabletible to degradation caused by an acidic environment 1) carry free carboxylic end groups, we suspecteddue to its linear character [17]. Therefore, salmon that this might enhance DNA degradation due toDNA was chosen as a model compound to test the acidification of the DNA-containing aqueous phaseinfluence of the type of polymer and various para- while forming the W/O dispersion. We thereforemeters on the encapsulation of DNA. For microen- determined the pH of the aqueous phase aftercapsulation, a W/O dispersion consistent of polymer / formation of the W/O dispersion and subsequent


Fig. 2. (continued)


Fig. 2. (continued)

phase separation by centrifugation with selected ing their loading efficiency and release kinetics (Fig.PLGA polymers. Interestingly, with all polymers a 7A and B). Higher loading was achieved with thesignificant drop in pH was observed which reached more hydrophilic polymers, including those con-values as low as pH 3.5 (Fig. 5). The drop of pH was taining a higher fraction of glycolide monomer andmost pronounced by using ethyl formate, which is those bearing uncapped end groups (Fig. 7A:probably due to rapid degradation of the solvent. The RG502H versus RG502, RG503H versus RG503,addition of 0.1 M NaHCO to the aqueous phase and R202H versus R202). Furthermore, higher vis-3

prior to formation of the dispersion prevented the cosity of the polymer containing phase (Fig. 7A:previously observed acidification, and the pH re- high versus low) and higher molecular weight (Fig.mained stable around 7.4 (Fig. 5). Indeed, micro- 7A: RG503H versus RG502H and RG503 versusspheres formed in the presence of NaHCO con- RG502) resulted in increased loading efficiencies,3

tained a significantly higher amount of dsDNA, as which may be due to faster formation of a solidshown before by PicoGreen analysis [17]. microsphere keeping the DNA entrapped.

Microspheres loaded with high amounts of DNAresulted in slightly larger and somewhat wrinkled 3.5. In vitro DNA release from microspheresmicrosphere surface, with the majority of the par-ticles still being ,10 mm in diameter (Fig. 6). A In the typical release pattern of hydrophilic mole-possible explanation for this phenomenon may be the cules encapsulated by biodegradable PLGA, twohigh viscosity of the aqueous phase which may have phases can be distinguished: the initial burst releasecaused a slower evaporation of the fluids during the and the second release phase which is due to gradualspray-drying process. Rose Bengal partitioning degradation of the polymer [4,17]. Selected micro-among different PLGA-type microspheres displayed sphere formulations were tested for the release ofa similar character with plain and DNA-loaded dsDNA in vitro. The burst release was found to be inmicrospheres. However, higher adsorption of the dye the range of 15–35% (Fig. 7B), whereas a significantoccurred to the enlarged surface of the wrinkled second release of dsDNA within 6 weeks of incuba-DNA-loaded microspheres (Figs. 1 and 6). tion was only detected with microspheres prepared

Microspheres generated with different polymer from the more hydrophilic polymers RG502H andformulations displayed significant differences regard- RG502 (Fig. 7C).


Fig. 3. Micrographs of DCs after phagocytosis of RG502H microspheres (30 min). (A) Sections of epon embedded DCs with phagocytosedparticles (stars) viewed by light microscopy (bar520 mm). (B) Transmission electron micrograph of a DC containing a microsphere (bar52mm). (C) The phagocytosed particle is tightly surrounded by a phagosomal membrane (arrows) (bar51 mm).


Fig. 4. (A) Degradation of fluorescent-labeled PLGA (RG502H) microspheres in MFs over time. (B) Different fluorescent-labeled PLGAmicrospheres 13 days after uptake by MFs. Cells were examined simultaneously by phase contrast (left panel) and fluorescence microscopy(right panel). Bar520 mm.

3.6. Microencapsulation of plasmid DNA However, the fate of small size microspheres afterphagocytosis by MFs might be quite different as

Based on our previous results, the formulation they end up in phagosomes known to acidify to pHRG502H/high was chosen to further encapsulate values down to 5.4 [36–38]. This may have an effectplasmid DNA. The encapsulation efficiency was on the release of encapsulated material since thecomparable to that of salmon DNA with 5665% degradation of PLGA polymer is triggered by thetotal DNA (determined by the A method) and cleavage of ester bonds which would be expected to260

57613% double-stranded DNA (determined by the be enhanced at lower pH [39]. We therefore followedPicoGreen method). Plasmid DNA extracted from the in vitro release of plasmid DNA from micro-the microspheres maintained most of its supercoiled spheres at different pH conditions from pH 7.4 to pHform as demonstrated by agarose gel electrophoresis 5.4. The burst release was in the range of 10–12%(Fig. 8A). and was similar under all pH conditions (Fig. 8B).

In general, in vitro release studies of PLGA DNA released after 1 day predominantly occurred inmicrospheres are performed at pH 7.4 [23,24,35]. the open circular form (Fig. 8A) and maintained a


Fig. 4. (continued)


Table 2Viability of cells after phagocytosis of microspheres (PLGA-6-coumarin)

bAmount of particles Day Mean fluorescence of % of all gated cellsa,bapplied to cells all gated cells (arbitrary units)

c c6-coumarin Apoptosis Necrosis

Control 1 – 4.3 (1.3) 7.6 (3.4)10 mg/well 1 2.9 (0.4) 4.3 (1.6) 5.6 (1.1)100 mg/well 1 45.4 (7.0) 3.5 (2.0) 6.8 (2.7)Control 7 – 3.8 (1.8) 9.6 (4.6)10 mg/well 7 2.7 (0.7) 1.9 (0.4) 9.4 (3.6)100 mg/well 7 23.8 (5.0) 1.1 (0.2) 7.4 (1.8)

a Corrected for background fluorescence (control cells).b Data are presented as mean (S.D.) for n53–4.c Apoptosis5Cells staining positive with annexin V-phycoerythrine but negative with propidium iodide; necrosis5all cells staining

positive with propidium iodide.

transfectivity of about 50% (Table 3). The topo- in accordance to enhanced particle degradation inlogical form of control DNA incubated under identi- cell culture studies.cal conditions for 1 day was not affected (Fig. 8A).Prolonged incubation of control DNA for 2 weeksresulted in a significant loss down to about 30% of 4. Discussionits biological activity and was comparable for all pHconditions (Table 3). DNA released from micro- The encapsulation of therapeutic compounds intospheres maintained significant biological activity small size biocompatible and biodegradable micro-even when microspheres were incubated at acidic spheres represents an attractive tool to targetconditions as low as pH 5.4 (Table 3). Acidic phagocytic cells such as DCs and MFs. The de-conditions enhanced DNA release (Fig. 8B) which is velopment of such delivery systems requires the

investigation of target cell interactions and further-more, the behavior of the encapsulated compound toidentify a suitable carrier material in concurrencewith the encapsulation process. Here we compare theproperties of different PLGA microspheres for theircapabilities to interact with human APCs in vitro,and disintegrate inside these cells. We have placedspecial emphasis on DCs and on fast degradingmicrospheres which are able to release their contentsintracellularly during the life span of the cells. AsDNA has become more important for vaccinationpurposes, we checked different PLGA type polymersfor their suitability to encapsulate and release DNA.

Surface hydrophobicity has been reported to beone of the major factors regulating phagocytosis ofmicrospheres by phagocytic cells [36], with hydro-phobicity favoring uptake [36]. We have tested

Fig. 5. Influence of polymer type and solvent on the pH of the microspheres composed of different PLGA typeDNA-containing aqueous phase after forming a W/O dispersion

polymers representing a range of surface hydro-by means of ultrasonication (20 s). Control values represent thephobicities in human APCs. To our surprise, all theseaqueous phase without adding polymer solution. Data are repre-

sented as mean for n52. different PLGA preparations were phagocytosed by


Fig. 6. Scanning electron micrographs of typical microspheres from different PLGA type polymers with 0.2% and 2% nominal loading ofDNA. Bar510 mm.


Fig. 7. DNA loading (A) and DNA release (B, C) from microspheres formed by different PLGA polymers. The nominal loading of DNAwas 2% and DNA was dissolved in 0.1 M NaHCO prior to encapsulation. The polymers were dissolved in methylene chloride resulting at3

low and high viscosity. Thereby, PLGA (14 000 and 17 000) are used at concentrations of 5% (low) and 10% (high) and PLGA (35 000) areused at concentrations of 2.5% (low) and 5% (high) to achieve comparable viscosity with polymers differing in molecular weight.Encapsulation efficiency was determined for the total of DNA (A ) and double-stranded DNA (PicoGreen). Release studies were260

performed in PBS at pH 7.4 and samples were analysed for double-stranded DNA. Data are represented as mean (6S.D. in A and C) forn53.

the cells to very similar extents. DNA loading of the and necrotic cell fragments, yeast, and bacteria [40–microspheres did not affect the phagocytosis by 44]. However, comparisons between DCs and MFseither MFs or DCs (data not shown). Even the are scarce and suggested that the phagocytic capacitymicrospheres prepared from most hydrophilic poly- of DCs would be inferior to that of MFs in vitro andmer RG502H were readily internalized by both DCs in vivo [40,43]. To our knowledge, this is the firstand MFs. We therefore conclude that all PLGA study that shows uptake of PLGA microspheres bymicrospheres tested in our study display a surface DCs and compares the uptake efficiency with highlyhydrophobicity in the range of optimal uptake. phagocytic macrophages. We found a significant

Immature DCs very efficiently capture soluble uptake of PLGA microspheres by DCs, whichantigens, but they have also been shown to reached 50–75% of the phagocytosis efficiency ofphagocytose particles such as latex beads, apoptotic MFs. The localization of microspheres inside


Table 3Transfectivity of plasmid DNA released from RG502H micro-spheres

aTime Transfected cells (% of control)

pH 7.4 pH 6.2 pH 5.4

Day 1 52.0 (4.6) 51.7 (3.7) 43.1 (3.8)Day 9 n.d. n.d. 49.0 (7.5)Day 12 n.d. 31.7 (2.1) 23.7 (1.9)Day 16 n.d. 74.6 (4.6) 50.3 (3.9)Day 20 n.d. 61.8 (16.4) 11.3 (2.3)Day 23 38.4 (2.7) 14.6 (1.7) 12.7 (1.3)Control day 23 32.3 (7.3) 28.4 (6.8) 30.5 (6.4)

a Transfectivity was tested in 293 cells after complexation ofDNA with Superfect and compared with untreated control DNA

based on the amount of double-stranded DNA detected by thePicoGreen assay. Control day 23 represents DNA incubated at378C under identical conditions as applied in the release experi-ments. Data are represented as mean (S.E.) for n53; n.d., notdetermined.

lated material at low pH ([39], this study). Inaddition, we speculate that lysosomal enzymes,especially those with esterase activity, increase therate of hydrolysis of PLGA polymers [45,46]. Lipidsand other biological components, present in thephagocytic compartment, may contribute to micro-sphere decay in yet another way: these may act asFig. 8. Encapsulation of plasmid DNA in RG502H/high micro-plasticizers that increase chain mobility and favor thespheres. The nominal loading of DNA was 2% and DNA wasuptake of water into the polymer [47].dissolved in 0.1 M NaHCO prior to encapsulation. (A) Agarose3

gel electrophoresis of DNA extracted and released (day 1) from None of the microsphere preparations significantlymicrospheres: lane 1, l DNA/Hind III ladder; lane 2, plasmid affected viability in exposed DCs or MFs, evenDNA control; lane 3, DNA extracted from microspheres; lane 4,

when the cells were loaded with very large numberscontrol pH 7.4; lane 5, released pH 7.4; lane 6, control pH 6.2;of microspheres. Cells maintained their viability alsolane 7, released pH 6.2; lane 8, control pH 5.4; lane 9, released pHduring microsphere decay which results in the re-5.4. (B) DNA release from microspheres dependent on the pH of

the release medium. Release studies were performed in PBS at pH lease of biocompatible but rather acidic degradation7.4, pH 6.2 or pH 5.4 and samples were analysed for double- products.stranded DNA. Data are represented as mean for n53.

In a previous paper, we have investigated thefeasibility of DNA encapsulation by one PLGApolymer (RG502) ([17], Table 1). Here we compare

phagosomal vesicles of DCs was clearly demon- the properties of different PLGA formulations instrated by electron microscopy. their capabilities to encapsulate and release DNA.

Phagocytosed microspheres remain inside the cells Our results show that the molecular weight anduntil decay, which was complete after 9 to 13 days hydrophobicity of the polymer are important deter-for the most hydrophilic polymer RG502H. Thus, minants for both the encapsulation and the release ofintracellular decay was enhanced compared to in DNA from spray-dried microspheres. In addition tovitro release studies in PBS. We believe that this may increased viscosity of the organic phase [18,23],be due in part to the acidification of the phagocytotic more hydrophilic polymers also displayed highercompartment, in analogy with enhanced in vitro encapsulation efficiencies. We suggest an emulsify-degradation of PLGA and faster release of encapsu- ing effect of hydrophilic polymers which results in


increased stability of the W/O emulsion and which is of encapsulated DNA, could enhance gene transfer tofurther enhanced by buffered aqueous solutions. APCs, because the quick release reduced the decay

Deleterious acidification of the DNA containing of DNA inside the microspheres before the deliveryaqueous phase already during the encapsulation of the genetic information. Moreover, the earlyprocess was observed with all PLGA formulations availability of DNA from RG502H microspheres andtested. However, by adding NaHCO to the aqueous the lack of toxicity in the target cell would prolong3

phase, we were able to improve both the incorpora- the time span during which gene expression couldtion and the stability of DNA in RG502 micro- take place. The approaches taken by Tinsley-Brownspheres [17]. A similar approach was reported by et al. [18], who optimized water-in-oil-in-waterShao and Bailey [48] where the incorporation of double emulsion process to produce hollow particlessodium bicarbonate in PLGA microspheres resulted with the encapsulated DNA in the center cavity, andin an improved release profile of porcine insulin. by Singh et al. [25], who employed cationic mi-Alternatively, protection of DNA was achieved by croparticles in combination with a cationic surfactantcomplexation to poly-L-lysine prior to encapsulation with the DNA adsorbed to the particle surface,[49–51]. In this formulation DNA was released from indicate an alternative prospect how DNA releasethe microspheres in complexed form with a sig- from PLGA microspheres can be accelerated.nificantly delayed second release phase [51]. In this study we were not able to monitor signifi-

The initial burst release was in the range of 15– cant reporter gene expression in MFs and DCs,35% and was comparable for all formulations. The although a sufficient number of microspheres werecontact with water possibly induced an initial swell- phagocytosed by these (data not shown). This coulding of the polymer at the particle surface and DNA be due to limited access of the DNA to the cyto-located close to the surface of the microspheres was plasm and/or nucleus or due to insufficient sensitivi-released during the first 24 h. Microspheres com- ty of the reporter gene assay [52]. Cytoplasmicposed of hydrophilic polymers like RG502H released delivery of intact DNA is important to ensure accessDNA faster during the second release phase. In this to the nucleus and subsequent synthesis of thecontext, our data showing DNA release in capped encoded protein. As we have demonstrated thatand uncapped PLGA polymers are in good accord- biologically active plasmid DNA is released fromance with the time dependence of water uptake RG502H microspheres even under slightly acidicreported by Tracy et al. [35]. When comparing conditions, transfer of DNA from the phagosomes touncapped PLGA to the more hydrophobic capped the cytosol of the target cells is suggested topolymer, initial degradation of the uncapped polymer represent a critical issue. Recent results reveal cyto-occurred earlier [35], presumably due to faster water plasmic delivery of macromolecules encapsulateduptake triggered by the hydrophilic end groups in the into PLGA microspheres after phagocytosis byuncapped polymer. This is an important factor, macrophages [53]. Moreover, our delivery systembecause we demonstrated that faster release of DNA offers the advantage of co-encapsulating agentsresulted in higher amounts of dsDNA released during which will stabilize DNA against enzymatic degra-the second release phase. dation, such as already demonstrated by Capan et al.

The fact that intact plasmid DNA was released [49,50], and which can enhance phagosomal escapefrom RG502H microspheres, may have been also by being released from microspheres together withdue to a higher intraparticular fluid exchange neutral- the DNA.izing some of the acidic compounds released during In conclusion, we have identified a hydrophilicpolymer degradation, thus reducing DNA decompo- PLGA polymer as a suitable material for the target-sition [17,35]. In our previous report using the more ing of microencapsulated therapeutics to MFs andhydrophobic RG502, we were not able to detect DCs. Microspheres prepared with the hydrophilicintact plasmid DNA released during the second PLGA polymer RG502H display improved releaserelease phase [17]. properties, coupled to high phagocytosis rates in

We suggest that more hydrophilic polymer formu- MFs and DCs, and do not affect the viability oflations, such as RG502H, which favor rapid release these cells despite their rapid degradation into acidic


building better vaccines, Nat. Biotechnol. 16 (1998) 1025–hydrolysis products. Furthermore, enhanced water1031.uptake into RG502H microspheres may lead to

[8] E. Mathiowitz, J.S. Jacob, Y.S. Jong, G.P. Carino, D.E.improved stability of the encapsulated material due Chickering, P. Chaturvedi, C.A. Santos, K. Vijayaraghavan,to a more efficient exchange of otherwise acidified S. Montgomery, M. Bassett, C. Morrell, Biologically erod-intraparticular fluid. Future experiments will be able microspheres as potential oral drug delivery systems,

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hydrophilic poly(dl-lactide-co-glycolide) microspheres for the delivery of dna to human-derived...

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