Hyaluronan microspheres for sustained gene delivery and site-specific targeting

ARTICLE IN PRESS

Biomaterials 25 (2004) 147–157

Hyaluronan microspheres for sustained gene delivery and site-specific targeting Yang H. Yuna, Douglas J. Goetzb, Paige Yellena, Weiliam Chena,* a

Department of Biomedical Engineering, State University of New York, 348 Psychology A Building, Stony Brook, NY 11794-2580, USA b Department of Chemical Engineering, Ohio University, Athens, OH, USA Received 21 December 2002; accepted 15 June 2003

Abstract Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5–20 mm. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hyaluronic acid/hyaluronan; Microsphere; DNA; Gene transfer; Selectin

1. Introduction Hyaluronan (HA) is a naturally occurring glycosaminoglycan distributed throughout the extracellular matrix, connective tissues, and organs of all higher animals [1,2]. The structure of HA consists of repeating disaccharide units of d-glucuronic acid and (1-b-3) Nacetyl-d-glucosamine [3–5]. The molecular weight typically ranges from 1
105 to 5
106 Daltons [5,6]. The importance of HA is evident by its key roles in the structure and organization of the extracellular matrix [7], transport of nutrients, regulation of cell adhesion [8,9], morphogenesis [10], and modulation of inflammation [11]. Since HA is native to the body, it is non-immunogenic and could be an ideal biomaterial for tissue engineering [12,13] and drug and gene delivery [14,15]. To date, HA has been successfully utilized for biomedical applica*Corresponding author. Tel.: +1-631-689-1593, +1-631-6890200x2247; fax: +1-631-632-8577. E-mail address: [email protected] (W. Chen). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00467-8

tions as hydrogels for viscosurgery and viscosupplementation [16,17], scaffolds for wound healing applications [18], and hydrophilic coatings for medical devices [19]. For drug and gene delivery applications, polymeric microspheres offer flexibility in dosing, release kinetics, and receptor targeting. However, the number of published methodologies for formulating HA microspheres is limited, and no formulations of microspheres using native HA for the purpose of long-term delivery of macromolecules (e.g., DNA) have been previously reported. In this article, we describe a novel method for preparing HA microspheres by adapting a non-toxic and aqueous based crosslinking chemistry. The derivatization chemistry of HA utilizing adipic dihydrazide has been used to construct hydrogels (see Fig. 1) [1,19] but never applied for microsphere preparation. Formulating HA microspheres with this method have several advantages. First, HA derivatives are well defined [20,21], the structural integrity and molecular size of HA are unaffected by the mild reaction conditions [1], and the by-products of the crosslinking

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Hyaluronan O

(

H

O

OH C

H

HO

H

O

HO H

O

OH H

H

H

H

O O

HO

NH H

H

H

C

H

OH H H

O

H

H H

O

NH2-NH

C

C

NH-NH2

O

N

+ HCl

N C N

ADH

EDCI

crosslinked Hyaluronan

( (

H

NH C

H

HO

H3C

H

H

HO

OH H

H

H

O

H

H

O O

NH H

H

HO H

C

O

H

O

H H

NH

CH 3

C

H

H O

H

HO

OH

O

H

NH O

O

NH NH

H C

H C

H

OH

NH

OH H

O

NH

O

H

H

NH

NH O

H OH

H

O

H

H

O

) ) O

O

OH

O

HO

H

O

NH

C

NH

H

O

CH 3

NH H

HO

O

C

O

O

H

OH H H

C

O

O C

OH

H

O

O

OH

OH

) O

H

HO

O

C

O

NH H

CH 3

O

O

CH 3

C

OH

H

C

H

OH

H

H

OH H

C CH 3 O

C O

Fig. 1. Schematic of chemical reaction for crosslinking of HA polymer with adipic dihydrazide. (adapted from Pestwich et al.) [1].

reaction such as urea and unreacted reagents can be easily removed by conventional methods such as dialysis, precipitation, and ultra-filtration. To demonstrate the utility of HA microspheres, we show that DNA can be easy incorporated before derivatization. Once the HA-DNA microspheres degrade, the released DNA is structurally intact and able to transfect cells in culture and in vivo using the rat hind limb model. Furthermore, HA microspheres coated with the antibody for E- and P-selectin exhibit selective adhesion to cells expressing these receptors [22–24], which are up-regulated in certain disease settings [25–27].

2. Materials and methods 2.1. Preparation of hyaluronan microspheres HA-DNA microspheres were prepared by a modified in-emulsion crosslinking technique. A water-in-oil emulsion was formed by homogenizing 20 ml of 0.5%

hyaluronan (1.6–3.3
106 Daltons, Kraeber GMBH & Co., Waldhofstr, Germany) solution with 100 mg of dissolved adipic dihydrazide (ADH, Sigma-Aldrich, St. Louis, MO), 80 ml of mineral oil (Sigma-Aldrich, St. Louis, MO), and 1 ml of Span 80 (ICI Chemicals, Wilmington, DE). This solution was mixed for 30 min at 1000 RPM using a mechanical stirrer (LR400D Lab Stirrer, Fisher Scientific, Pittsburgh, PA) fitted with a 0.75 in diameter impeller (Cole-Parmer, Vernon Hills, IL). A solution of plasmid DNA (pDNA) encoding a bgalactosidase (b-gal, BD Biosciences Clonetech, Palo Alto, CA) reporter regulated by the cytomegalovirus immediate early promoter was added to the emulsion. To ensure even distribution of pDNA into the aqueous phase, the emulsion was stirred for an additional 20 min. The pDNA loading was 1 or 5 mg (1% and 5%, respectively). For plain HA microspheres, this step was omitted. Afterwards, 120 mg of ethyl-3-[3-dimethyl amino] propyl carbodiimide (EDCI, Sigma-Aldrich, St. Louis, MO) dissolved in 2 ml of distilled and deionized water (DH2O) was slowly instilled and mixed for 30 min.

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The addition of hydrochloric acid (HCl, 0.1 n, 0.3 ml) subsequently initiated the crosslinking of the HA microspheres. The chemical reaction, at room temperature, was allowed to proceed for 24 h. The HA-DNA microspheres were precipitated by the addition of 150 ml of isopropyl alcohol (IPA) under vigorous agitation. The aqueous layer, after separation from the mineral oil, contained the HA-DNA microspheres and was isolated. HA-DNA microspheres were centrifuged at 1500 RPM for 5 min. The supernatant was discarded and the microspheres were washed three times by centrifugation (1500 RPM for 5 min) with IPA. After the final wash, the microspheres were resuspended in a reagent mixture containing 100 mg of ADH and 120 mg of EDCI dissolved in 100 ml of 90% dimethylformamide (DMF) or 90% IPA. This mixture was gently stirred using a stir bar and a magnetic plate. HCl (0.1 n, 0.3 ml) was subsequently added to initiate the second crosslinking reaction. After 24 h, the microspheres were collected by centrifugation and washed 3 times with 90% IPA. They were then resuspended in distilled water, frozen on dry ice, and lyophilized (Virtis Freezemobile 12EL, Gardener, NY).

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tries, Berkeley, CA). After 6 h, HA-DNA microsphere suspensions were centrifuged at 3000 RPM and 0.9 ml of the supernatants was collected. The HA-DNA microspheres were resuspended in an equal volume of fresh hyaluronidase solution. Additional DNA samples were collected at pre-determined time points (terminated at 63 days). As controls, HA-DNA microspheres were dispersed in PBS containing no hyaluronidase and the released DNA was collected as described. A PicoGreens (Molecular Probe, Eugene, OR) fluorometric assay [28] was used to determine the pDNA concentration of the supernatants collected during the course of the release studies. Briefly, PicoGreens was diluted 200 fold with Tris–EDTA (TE, Sigma-Aldrich, St. Louis, MO) buffer. The diluted PicoGreens reagent (50 ml) was mixed into equal volumes of the DNA sample and incubated at room temperature for 30 min. The samples’ fluorescent signals were determined with a cytofluorometer (CytoFluor 4000, PerSeptive Biosystem, Framingham, MA) at an excitation wavelength of 480 nm and emission wavelength of 530 nm. The DNA contents in the samples were determined by referencing the fluorescent signals against a standard plot prepared previously.

2.2. SEM of HA microspheres 2.5. Gel electrophoresis analysis The structural conformation of HA-DNA microspheres was observed using a scanning electron microscope (SEM, Joel 5300, Peabody, MA). To prepare for SEM, microspheres were initially suspended in DH2O. After they were dispersed, the HA-DNA microspheres were dehydrated in a series of ethanol solutions (20%, 40%, 60%, 80%, 90%, 95%, 100%) and then sputter coated with gold/palladium. 2.3. Size distribution of HA microspheres Approximately 100 mg of HA-DNA microspheres was suspended into 10 ml of phosphate buffered saline (PBS, pH 7.4, Sigma-Aldrich, St. Louis, MO, sterilized with 0.2 mm filter). After an hour of equilibration in PBS, the size distribution of HA-DNA microspheres was determined with a laser scattering particle size analyzer (LA-190, Horiba Ltd., Irvine, CA). 2.4. Release kinetics of pDNA from HA-DNA microspheres The DNA release kinetics were evaluated for HADNA microspheres with 1% and 5% DNA loading (the percentage of DNA added to the HA solution during the formulation). The HA-DNA microspheres were dispersed 1 ml of hyaluronidase (HAse, Sigma-Aldrich, St. Louis, MO) solution (10 units/ml in PBS). They were then incubated at 37 C under constant agitation using a rotary agitator (LabQuake shaker L-1237, Lab Indus-

Electrophoretic motility analyses were performed on the DNA release samples collected during the release kinetics study. Agrose gel (1%, BioWhittaker Molecular Applications, Rockland, ME) containing ethidium bromide (0.5 mg/ml, Fisher Scientific, Pittsburgh, PA) was cast and loaded with the DNA samples collected during the release study. For comparisons, HA-DNA microspheres with 1% and 5% DNA loading were dispersed in TE buffer, and HA solutions mixed with DNA (not crosslinked, 1% and 5% loading) were also analyzed. 2.6. In vitro cellular transfection The bioactivity of the pDNA recovered during the course of the release kinetics experiments was assessed with a gene transfer study using Chinese Hamster Ovarian (CHO, ATCC, Rockville, MD) cells. Cells were plated onto 24 well tissue culture plates at a density of 20,000 cells/well and maintained with growth media that consisted of Dulbecco Modified Eagle Medium (DMEM, Cellgro, Heindon, VA) with 10% fetal calf serum (Cellgro, Heindon, VA). After cells were grown to approximately 60% confluency, the growth media was replaced with a transfection reagent mixture consisting of 100 ml of DNA samples (collected during the course of the release kinetics study), 100 ml of LipofectAMINEt (Gibco Invitrogen, Carlsbad, CA), and 300 ml of DMEM. The CHO cells were incubated in the

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Y.H. Yun et al. / Biomaterials 25 (2004) 147–157

LipofectAMINEt/DNA mixture for 4 hours, and the transfection reagent was replaced with growth media. After 48 h, the CHO cells were fixed with 1% formaldehyde in PBS and cytochemical analysis (X-Galt staining, Gibco Invitrogen, Carlsbad, CA) was performed thereafter. Cells were immersed in a X-Galt solution (1 mg/ml, 5-bromo-4-chloro-3-indolyl-b-d-galactoside, 5 mm K3[Fe(CN)6], 5 mm K2[Fe(CN)6], 2 mm MgCl2, in PBS, pH 7.4) and incubated overnight at 37 C. The number of cells expressing b-gal was manually counted and their relative levels of transfection were reported. 2.7. In vivo rat hind limb transfection All animals received humane care in compliance with the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). Furthermore, the Institutional Animal Care and Use Committee for the State University of New York at Stony Brook reviewed and approved the protocol used in this study (IACUC #2002-1074). HA-DNA microspheres (loaded with pDNA encoding for b-gal) were injected into the hind limb muscles of rats and evaluated for their transcription into messenger RNA (mRNA). Ketamine (40–80 mg/kg) and Xylazine (5–10 mg/kg) were injected into the intrapleural cavity. Once the rats were anesthetized, their hind limbs were shaved and aseptically prepared for microsphere injection. The injection spot was tattooed to mark the site of injection and for a reference when harvesting. HA-DNA microspheres were reconstituted (10 mg with 1% DNA loading) with sterile saline (50 ml), loaded into a 0.5 cm3 insulin syringe (BD, Franklin Lakes, NJ), and injected into the hind limb muscle at the tattooed site. The positive and negative controls were injected on the contra-lateral hind limb of the rats. For positive control, polylactic-co-glycolic acid (PLGA, 6000 MW, 50:50 lactic to glycolic acid ratio) microspheres were prepared using an in-emulsion solvent evaporation technique as previously described [29]. PLGA microspheres were reconstituted in sterile saline before injection. The dosage of PLGA microspheres (10 mg with 1% DNA loading) was matched to the dosage of HA microspheres. For the negative control, 50 ml of saline was injected. Three weeks after the injections, rats were placed into a CO2 gas chamber and euthanized. The hind limb muscles were promptly harvested and frozen in liquid nitrogen. This entire procedure, from euthanasia to harvesting, was conducted in less than 5 min per rat to ensure the RNA’s integrity. The frozen muscles were

Table 1 Sequence of b-gal primers used for the RT-PCR studies Direction

Sequence

Forward Backward

50 -ACCCGCATTGACCCTAAC-30 50 -TGTATCGCTCGCCACTTC-30

crushed into a powder in the presence of liquid nitrogen using mortar and pestle, transferred into RNase free centrifuge tubes containing Trizols (Gibco Invitrogen, Carlsbad, CA), and homogenized (PowerGen 700, Fisher Scientific, Pittsburgh, PA). Total RNA was then extracted according to the manufacturer’s instructions. The precipitated RNA was washed, suspended in RNase free water, and stored at 80 C until purification. DNA and pDNA contaminates were digested before purification of the extracted RNA. RNA (200 ng) was incubated with DNase I (1 U/ml, RNase free, F. Hoffmann-La Roche Ltd, Basel, Switzerland) for 15 min at 25 C. Using the RNA cleanup procedure from the RNeasys kit (Qiagen Inc., Valencia, CA), RNA was purified and stored at 80 C until analysis. To determine if the muscle tissue was transcribing the delivered pDNA encoding for b-gal, the extracted RNA was analyzed with reverse transcriptase polymerase chain reaction (RT-PCR) with primers shown in Table 1. One step RT-PCR using a kit (Qiagen Inc., Valencia, CA) was performed according to the manufacturer’s instructions. Thirty cycles were used for the denaturing (94 C for 1 min), annealing (53 C for 30 s), and the extension (68 C for 1 min) steps. The RT-PCR products were analyzed with gel electrophoresis using 1% agarose. The amplicons (234 base pairs) were sequenced to ensure it was from a b-gal origin. 2.8. In vitro adhesion of ligand coated HA microspheres Human Umbilical Vein Endothelial Cells (HUVECs, Clonetics, San Diego, CA) were maintained in culture as previously described [22,30]. To induce the expression of E-selectin, HUVECs were treated for 4 h with 50 U/ml of Interleukin-1b (IL-1b; Calbiochem, San Diego, CA) [31]. Chinese hamster ovarian (CHO) and CHO stably expressing human P-selectin (CHO-P) cells, which have been previously generated and characterized, were generous gifts from Dr. Raymond T. Camphausen (Wyeth Research; Cambridge, MA) [32]. The CHO and CHO-P cells were maintained in culture as previously described [22]. HuEP5C7.g2, a humanized monoclonal antibody (mAb) to E- and P-selectin (HuEP, a gift from Protein Design Lab., Fremont, CA) [33], was conjugated to the HA microspheres (HuEP HA microspheres) via protein A for selective cellular targeting. These microspheres were then perfused over cellular substrates under in vitro

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3. Results 3.1. Characterization of HA microspheres The water-in-oil emulsion of HA, DNA, and mineral oil that was crosslinked with adipic dihydrazide produced microspheres with spherical conformation and heterogeneous size distribution (see Fig. 2). The median and mean diameters of the HA-DNA microspheres were 6 and 20 mm, respectively, as indicated by a

Fig. 2. SEM of HA-DNA microspheres. The diameter of microspheres ranged from 5 to 15 mm.

10 8

Frequency [% ]

flow conditions that mimic, in part, flow conditions present in vivo. HuEP or human IgG (negative control, Sigma-Aldrich, St. Louis, MO) were coupled to the HA microspheres via protein A using a technique similar to that previously described for polystyrene [23] and poly(e-caprolactone) [24] microspheres. Briefly, HA microspheres were incubated with protein A (300 mg/ ml, Zymed, San Francisco, CA) for 1 h at room temperature. Subsequently, the microspheres were washed with blocking buffer HBBS+(Hank’s Balanced Salt Solution, supplemented with 1% BSA, Biowhittaker, Walkersville, MD) and incubated in blocking buffer containing 50 mg/ml HuEP or 50 mg/ml human IgG for 1 h. Following the incubation, the microspheres were washed and held in blocking buffer until used in the flow chamber assay. A parallel plate flow chamber (Glycotech, Rockville, MD), similar to that described by McIntire, Smith and colleagues [34], was used to study the adhesive properties of the ligand coated HA microspheres. Our particular set up has been described previously [35]. The temperature was maintained at 37 C with a heating plate. Briefly, a 35 mm tissue culture dish containing a cellular monolayer (i.e. HUVEC, IL-1b activated HUVEC, CHO or CHO-P) was loaded into the flow chamber. The flow chamber was mounted on an inverted microscope (Nikon Eclipse TE300) equipped with a videocamera, monitor and VCR. A suspension of HA microspheres (5
105 in HBSS+, 0.5% BSA) was drawn over the cellular monolayer at 1.5 dyn/cm2. After 2.5 min of flow, the shear was reduced to 1.0 dyn/cm2, and the flow was maintained for an additional 2.5 min. Subsequently, the number of HA microspheres adherent to the cells was determined for eight different fields of view. These numbers were averaged and normalized to the area of the field of view. In certain cases, IgG and HuEP microspheres were sequentially perfused over the same cellular monolayer. In these instances, the IgG microspheres were always perfused prior to the HuEP microspheres, and the number of adherent IgG microspheres was subtracted from the number of adherent microspheres observed after perfusion with the HuEP microspheres to arrive at the number of adherent HuEP microspheres.

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6 4 2 0

0

1

2

3

4

8

13

23

39

68

Particle Diameter [ µm]

Fig. 3. Size distribution of HA-DNA microspheres prepared from adipic dihydrazide chemistry.

particle size analyzer. The size distribution, seen in Fig. 3, indicated that more than 60% of the HA-DNA microspheres were between 3 and 10 mm. 3.2. Release kinetics of DNA from HA-DNA microspheres The release profiles from HA-DNA microspheres, as shown in Figs. 4A and B, demonstrated an initial burst release, but the rate of DNA release moderated thereafter. This initial release was independent of the enzyme activity in the buffer and the amount of DNA loading. Afterwards, the HA-DNA microspheres steadily released DNA until the termination of the experiment. For a period of 63 days, approximately 610 ng (cumulative) of DNA per 100 mg of microspheres was released from HA-DNA microspheres (1% loading, see Fig. 4A) in HAse buffer. Thus, approximately 60% of the microspheres’ DNA contents were cumulatively released. As seen in Fig. 4B, microspheres with 5% DNA loading released approximately 5 times more

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Cumulative DNA Released

(ng of DNA/100 µg microparticles )

800

600

400

200

0 0

13

(A)

26

39

52

65

(A)

TIME ( Days )

Cumulative DNA Released

( ng of DNA/100 µg microparticles )

3500

2800

2100

1400

700 (B)

0 0 (B)

13

26

39

52

65

Time (Days)

Fig. 4. DNA Release profile of HA-DNA microspheres: (A) HADNA microspheres with 1% DNA loading (.) in 10 units/ml of HAse and (&) in PBS. (B) HA-DNA microspheres with 5% DNA loading (K) in 10 units/ml of HAse and (n) in PBS.

DNA as compared to microspheres with 1% DNA loading (approximately 3000 ng of DNA per 100 mg of microsphere) which corresponded to 60% of the microspheres’ DNA contents. Overall, HA-DNA microspheres incubated in PBS released approximately one-third of the DNA as compared to HA-DNA microspheres incubated in HAse solution regardless of the loading conditions. 3.3. Gel electrophoresis of DNA released from HA-DNA microspheres The structural conformation of the released DNA was analyzed by performing gel electrophoresis of samples collected during the course of the release study. As shown in Fig. 5A, lanes 1 and 2 were loaded the 1KB DNA marker and the pDNA used to prepare the

Fig. 5. Gel electrophoresis: (A) Released DNA samples. (1) DNA molecular weight marker, (2) DNA for fabricating HA-DNA microspheres, (3,4,5,6) DNA samples collected during the course of a release study, (7) HA-DNA microspheres with 1% DNA loading, and (8) HADNA microspheres with 5% DNA loading. (B) HA and pDNA mixtures. (1) DNA molecular weight marker, (2) HA-DNA microspheres with 1% DNA loading, (3) HA-DNA microspheres with 5% DNA loading, (4) HA solution containing 1% DNA, and (5) HA solution containing 5% DNA.

HA-DNA microspheres respectively. Lanes 3–6 were loaded with representative DNA samples collected during the release kinetics experiment. As indicated by the double bands on the gel, the DNA released from the HA-DNA microspheres was intact. However, an increased proportion of uncoiled DNA from the release studies was observed as compared to the stock DNA shown in lane 2. The florescence along the paths of supercoiled and uncoiled pDNA samples was probably HA-DNA fragments that were sufficiently small to migrate into the gel and appeared as diffused fluorescence around the DNA bands. The faint fluorescent observed in the areas below the supercoiled DNA (the lower bands), was likely the released DNA that was partially degraded. The residual fluorescence observed in the loading wells could be attributed to HA-DNA

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fragments released from the microspheres that were too large to migrate into the gel. In Lanes 7 and 8, two different HA-DNA microsphere preparations were loaded directly into the gel after dispersion in TE buffer. The intense fluorescence present in the loading wells indicated that DNA was trapped inside the microspheres. The diffused fluorescence seen in the corresponding locations of supercoiled and uncoiled DNA was due to small amount of DNA leached from the HADNA microspheres. Fig. 5B depicted the results obtained by performing gel electrophoresis on non-crosslinked HA/DNA mixtures and HA-DNA microspheres. Lane 1 was loaded with the DNA molecular weight marker. Lanes 2 and 3 were loaded with HA-DNA microspheres with 1% and 5% DNA loading, respectively. The wells that were fluorescent indicated that either DNA did not migrate out of the HA-DNA microspheres or the amount of leaching was undetectable. Lanes 4 and 5 were loaded with HA solutions containing 1% and 5% DNA (noncrosslinked), and fluorescent smears were observed along lanes in conjunction with the fluorescence of DNA inside the wells. Due to the higher DNA loading, the fluorescent intensity in lane 5 (5% DNA loading) was expectedly much greater than the fluorescent intensity in lane 4 (1% DNA loading). 3.4. In vitro transfection of CHO cells The relative level of transfection depicted in Fig. 6 was determined in CHO cells using the DNA released from a representative HA-DNA microsphere preparation with 1% DNA loading. This result indicated the

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released DNA from HA-DNA microspheres was bioactive. A relatively high level of transfection was initially observed, but gradually decreased with time. The level of cell transfection was generally in agreement with the DNA release profiles depicted in Fig. 5A. The lower levels of transfection observed towards the end of the study were attributed to the decreased amounts of DNA released from the HA-DNA microspheres or the loss of activity of the released DNA due to degradation. 3.5. In vivo transfection of rat hind limb muscles Three weeks after injection, the results (see Fig. 7) of the RT-PCR analysis showed positive signals for rat hind limb muscles injected with HA-DNA and PLGA microspheres. Lanes 3 and 4 were loaded respectively with amplicons from the hind limb tissues of two animals injected with HA-DNA microspheres. The amplicon in lane 5 (a positive signal was present though it was faint) was generated from the RNA of a muscle injected with PLGA microspheres loaded with pDNA encoding for b-gal. The hind limb muscle injected with saline showed no signal (lane 2), and the stock pDNA encoding b-gal, used as a template for RT-PCR, showed a positive signal (lane 6). As expected, the amplicon from the stock pDNA was the same size as the hind limb muscles transfected from HA-DNA and PLGA microspheres. These results confirm that all the amplicons were from b-gal origin and the injected tissues were transfected and transcribing the pDNA released from HA or PLGA microspheres.

Relative Level of Transfection

6 5 4 3 2 1 0 0

10

20

30

40

50

60

70

Time ( Days ) Fig. 6. Relative levels transfection of CHO cells from DNA samples collected from the release study. The transfection decreased with time and corresponded to the amount of DNA released from the HA-DNA microspheres.

Fig. 7. RT-PCR from rat hind limbs that have been injected with HADNA microspheres, saline, or PLGA. Tissues were harvested 3 weeks after injection. (1) 100 base pair ladder, (2) saline, (3 & 4) HA-DNA microspheres with pDNA encoded for b-gal from 2 animals, (5) PLGA microspheres loaded with DNA encoded for b-gal, (6) b-gal DNA template for positive control.

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3.6. Selective adhesion of HA microspheres to E- and P-selectin expressing cells As shown in Fig. 8A, HA microspheres coated with HuEP exhibited significant adhesion to 4 hour IL-1b activated HUVECs. The level of adhesion of the HuEP HA microspheres to IL-1b activated HUVECs was significantly greater than the level of adhesion of HuEP HA microspheres to unactivated HUVECs. The IgG HA microsphere adhesion to 4 hour IL-1b activated HUVECs was quite low and statistically similar to the 25 Microspheres / mm2

* 20

(A)

0

15 10

level of adhesion of IgG HA microspheres to unactivated HUVECs. Previous studies have established that HUVECs pre-treated with IL-1b for 4 h express significant levels of E-selectin while unactivated HUVECs express little, if any, E-selectin [31]. It should be noted that the HUVECs used in the present study expressed little, if any, P-selectin in response to IL-1b stimulation [53]. CHO-P cells were used to investigate the adhesion of the HA microspheres to cellular expressed P-selectin. As shown in Fig. 8B, HuEP HA microspheres exhibited significant adhesion to CHO-P. The level of adhesion was significantly higher than the level of adhesion of HuEP HA microspheres to CHO cells not expressing P-selectin. The level of adhesion of IgG HA microspheres to CHO-P cells was relatively low and comparable to HuEP HA microsphere adhesion to CHO cells. Combined, the data presented in this section clearly indicated that HA microspheres coated with a humanized mAb to E- and P-selectin exhibited selective adhesion to cells expressing E- and P-selectin.

5

4. Discussion and conclusions

Cells: A-HUVEC Ligand: gand: 140

HuEP P

U-HUVEC HuEP

A-HUVEC U-HUVEC IgG G

IgG G

*

Microspheres / mm2

120

(B)

100 80 60 40 20 0 Cells:

Ligand:

CHO-P

CHO

CHO-P

HuEP

HuEP

G IgG

Fig. 8. HA microspheres coated with a humanized mAb to E- and Pselectin exhibited selective adhesion to E- and P-selectin expressing cells. (A) HA microspheres were conjugated with HuEP or human IgG (negative control) and subsequently perfused over HUVEC or HUVEC pretreated for 4 h with IL-1b: Subsequently the number of adherent microspheres was determined. (B) Experiments similar to part A were conducted with CHO-P and CHO as the adhesive substrate. (Legend. Cells indicates which cells were used in the adhesion assay: A-HUVEC—4 h IL-1b activated HUVEC; U-HUVEC—unactivated HUVEC; CHO-P—Chinese hamster ovary cells stably expressing P-selectin; CHO—Chinese hamster ovary cells. Ligand indicates which type was coupled to the HA microspheres: HuEP—humanized mAb to E- and P-selectin; IgG—human IgG. po0:05 compared to HuEP HA microspheres over U-HUVEC or HuEP HA microspheres over CHO.)

Recent reports describe the usefulness of microspheres prepared with synthetic polymers as sustained gene delivery vehicles [36–38], yet a search of the medical literature reveals only few methodologies for preparing HA into microspheres. For these formulations, HA is either esterified, blended with other polymers, or exposed to elevated temperature for stabilization. The preparation of HA microspheres has been first described by Fidia Advanced Biopolymers wherein the carboxyl groups of the glucuronic acid moieties of HA are esterified [39]. Lim et al. has prepared microcapsules by complex coacervation using HA and HA blended with gelatin and acacia [40]. Microsphere preparations using water-in-oil emulsion in which native HA and HA blended with chitosan also have been described by Lim et al. [40,41]. In this investigation, we present a novel formulation of HA microspheres prepared from native HA. The HADNA microspheres, crosslinked with adipic dihydrazide, release bioactive DNA for months and are adaptable for targeting cellular receptors. The advantages for formulating HA microspheres with dihydrazide crosslinking chemistry are the following: (1) native HA is utilized (instead of modified HA as used by Fidia Advanced Biopolymers [39]), (2) the chemical reaction occurs at room temperature (in contrast to elevated temperature used by Lim et al. [40]), (3) the use of chemical reagents such as hexane, chloroform, methylene chloride, or glutaraldehyde is not needed (as described in Lim et al. [39,40]), and (4) complicated mechanical equipment is not needed.

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The crosslinking native HA using adipic dihydrazide chemistry has extended the release profile of HA-DNA microspheres from days to months when compared to HA microspheres prepared by heat precipitation without any chemically mediated crosslinking [40]. The results at the first two time points show that the amounts of DNA released are approximately equal for microspheres dispersed in HAse and PBS buffer. This initial release is likely the consequence of DNA bound on the surfaces of HA microspheres but not fully incorporated into its matrix. Since the initial release is mediated by diffusion instead of degradation, the amount of DNA release should be independent of DNA loading or the presence of HAse in the release medium. At the end of the release study, the HA-DNA microspheres in HAse solution release three times more DNA than microspheres in PBS alone. The microspheres incubated in PBS are degraded only by hydrolysis; thus, both the rate and amount of DNA release are expected to be significantly less than HADNA microspheres in HAse solution. When the DNA loadings are compared, the microspheres with 5% loading release approximately five times more DNA than the microspheres with 1% loading. Finally, the release profiles and the release capabilities for HA-DNA microspheres in PBS are similar to the results for PLGA microspheres (6000 MW, 50:50 lactic to glycolic acid ratio, 2% DNA loading), which has been prepared by in-emulsion solvent evaporation method and degraded in PBS, previously published by Wang et al. [29]. The results from the electrophoresis studies show that the dihydrazide crosslinking chemistry is not destructive to the incorporated DNA. As seen in Figs. 5A and B, the released DNA is structurally intact, and the results from the transfection experiments (see Figs. 6 and 7) indicate that the release DNA is bioactive. However, the increased proportion of uncoiled DNA could be attributable to the brief exposure to high speed stirring during their preparation or the agitation required during the release studies. Indicated by Fig. 5B, DNA is tightly associated with HA (possibly through extensive hydrogen bonding) as seen by the fluorescent smearing observed on lanes 4 and 5. Only the DNA molecules that bind to the HA of lower molecular weight range (HA is a mixture with heterogeneous chain length thus composed of various molecular weight HA) could migrate into the gel. This association could serve as a protective mechanism for DNA from degradation and account for the minimal DNA degradation observed in lanes 3–6 in Fig. 5A. The transfection studies of the released DNA, shown in Fig. 6, illustrate that the sustained release of DNA could result in sustained transfection. Transfection of cells in culture shows good agreement with the release of DNA from HA-DNA microspheres (see Fig. 5). As expected, the initial burst release of DNA results in a

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high degree of initial transfection. Although the level of transfection decreases with respect to time, the bioactivity of the released DNA is still measurable. A greater degree of transfection for these periods could be attained by re-administration. Multiple administrations of HA-DNA microspheres are an advantage over vectors such as viruses that have shown to trigger an aggressive immune response. Furthermore, viruses and other delivery vehicles such as naked DNA and polyelectrolytes have short persistency in vivo. The success of gene delivery vehicles for use in clinical applications will likely depend upon optimal release [42,43] with specificity instead of maximal gene transfer for a short duration. For HADNA microspheres, the dose and the duration of release could be easily controlled by adjusting the DNA loading, the extent of crosslinking, and the timing of administration. The controlled released characteristics exhibited by HA-DNA microspheres are quite desirable and evident in vivo. The HA-DNA microspheres injected into the hind limb of rats show positive DNA transcription after 3 weeks (see Fig. 7). These results confirm that the hind limb tissues have been transfected by the DNA, which is not native to the rat genome, released from HA-DNA microspheres. Therefore, strategies for gene therapy using HA-DNA microspheres could be tailored to pathologies of specific diseases. For example, HA microspheres with two distinct release profiles loaded with genes encoding for vascular endothelial growth factor and platelet derived growth factor respectively could be used to stimulate functional blood vessels for chronic ischemic tissues [42,44]. Over the past decades, the vascular endothelium has been shown to be heterogeneous in its expression of carbohydrate and protein moieties [45–47]. This property has led to the idea that discretely expressed moieties could be used for the delivery of therapeutic agents to a selective tissue [23,24,48–51]. Specific examples of such moieties are the glycoproteins E- and P-selectin, which are up-regulated at various sites of diseased tissue [25– 27]. The efficacy of HA microspheres as a delivery vehicle can be enhanced by incorporating this targeting element. HA microspheres coated with a humanized mAb to E- and P-selectin exhibit selective adhesion to cells expressing these receptors (Figs. 8A and B). The selectivity of the adhesion is quite striking with a greater than 40-fold increase in the adhesion to activated HUVECs by IL-b relative to unactivated HUVECs and a six-fold increase in the adhesion to CHO-P relative to CHO cells. In addition, the level of adhesion is significantly better than previously published data with HuEP coated poly(b-caprolactone) microspheres [24]. Indeed, significant levels of adhesion of HuEP HA microspheres are observed at a shear stress of 1.0 dyn/ cm2, which is physiologically relevant.

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Interesting, the selectivity of HuEP coated HA microspheres to HUVECs is substantially higher compared to the selectivity over CHO cells (i.e. 44-fold selectivity vs. six-fold selectivity). This difference could be attributed to the high level of non-specific binding of the HuEP HA microspheres for the CHO cells relative to the non-specific binding to the HUVECs. This finding underscores the fact that polymeric adhesion to cellular substrates is quite complex and a function of a variety of factors including shear [23], particle chemistry, particle size [35] and cellular surface chemistry. In regards to particle size, the diameter of HA microspheres used in this study is slightly above the size that could be introduced into the circulation. The upper size limit for use in vivo is B2 mm. For the HA microspheres to be endocytosed by endothelial cells, they should be less than 200 nm [52]. While we have established selective binding of the HuEP HA microspheres to selectin presenting cells, more work is needed to bring this targeting approach to fruition such as optimizing the HA properties to achieve maximal selective adhesion to the target segments of the endothelium. In conclusion, microspheres prepared with native HA can be crosslinked by adapting an adipic dihydrazide crosslinking chemistry, a method that has originally used to prepare HA into hydrogels. The resulting microspheres are biodegradable yet robust in an aqueous environment. As the microspheres degrade, the incorporated DNA releases for approximately 2 months. The released DNA is structurally intact and able to transfect cells in culture. More significantly, the HA-DNA microspheres are able to transfect skeletal muscle in vivo, which has been verified by RT-PCR, and are adaptable for targeting specific cellular receptors to distinguish E- and P-selectin expressing cells. These characteristics suggest that HA-DNA microspheres could an ideal vehicle for systemic delivery of therapeutic genes.

Acknowledgements This study was supported by a grant from the National Heart, Lung and Blood Institute (R01 HL65175; WC), and an individual grant from The Whitaker Foundation (DJG). The authors would like to thank Dr. Rajinder Bhasin of Corner Stone Pharmaceutical, Inc. for his assistance in performing particle size analysis and Ms. Amy Neuforth for data collection.

[2]

[3]

[4] [5] [6]

[7] [8]

[9]

[10] [11]

[12] [13]

[14] [15]

[16] [17]

[18] [19]

[20]

[21]

[22]

References [1] Prestwich GD, Marecak DM, Marecek JF, Vercruysse KP, Ziebell MR. Chemical modification of hyaluronic acid for drug delivery, biomaterials, and biochemical probes. In: Lauret TC,

[23]

editor. The chemistry, biology, and medical applications of hyaluronan and its derivatives. Miami, FL: Portland Press; 1998. p. 43–65. Fraser JR, Brown TJ, Laurent TC. Catabolism of hyaluronan. In: Lauret TC, editor. The chemistry, biology, and medical applications of hyaluronan and its derivatives. Miami, FL: Portland Press; 1998. p. 85–92. Goa KL, Benfield P. Hyaluronic acid. A review of its pharmacology and use as a surgical aid in ophthalmology, and its therapeutic potential in joint disease and wound healing. Drugs 1994;47:536–66. Laurent TC, Fraser JR. Hyaluronan. Faseb J 1992;6:2397–404. Toole BP. Hyaluronan and its binding proteins, the hyaladherins. Curr Opin Cell Biol 1990;2:839–44. Bertolami CN, Berg S, Messadi DV. Binding and internalization of hyaluronate by human cutaneous fibroblasts. Matrix 1992;12:11–21. Scott JE. Extracellular matrix, supramolecular organisation and shape. J Anat 1995;187:259–69. Hardwick C, Hoare K, Owens R, Hohn HP, Hook M, Moore D, Cripps V, Austen L, Nance DM, Turley EA. Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility. J Cell Biol 1992;117:1343–50. Collis L, Hall C, Lange L, Ziebell M, Prestwich R, Turley EA. Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action. FEBS Lett 1998;440:444–9. Toole BP. Hyaluronan in morphogenesis. J Intern Med 1997;242:35–40. Gerdin B, Hallgren R. Dynamic role of hyaluronan (HYA) in connective tissue activation and inflammation. J Intern Med 1997;242:49–55. Peppas NA, Langer R. New challenges in biomaterials. Science 1994;263:1715–20. Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK, Langer R. Biodegradable polymer scaffolds for tissue engineering. Biotechnology (New York) 1994;12:689–93. Langer R. Biomaterials in drug delivery and tissue engineering: one laboratory’s experience. Acc Chem Res 2000;33:94–101. Kim A, Checkla DM, Chen W. Characterization of DNAhyaluronan matrix for sustained gene transfer. J Control Release 2003;90:81–95. Balazs EA, Denlinger JL. Clinical uses of hyaluronan. Ciba Found Symp 1989;143:265–75. Davidson JM, Nanney LB, Broadley KN, Whitsett JS, Aquino AM, Beccaro M, Rastrelli A. Hyaluronate derivatives and their application to wound healing: preliminary observations. Clin Mater 1991;8:171–7. Juhlin L. Hyaluronan in skin. J Intern Med 1997;242:61–6. Luo Y, Kirker KR, Prestwich GD. Modifications of natural polymers: hyaluronic acid. In: Atala A, Lanza RP, editors. Methods of tissue engineering. San Diego, CA: Academic Press; 2001. p. 553–93. Pouyani T, Harbison GS, Preswich GD. Novel hydrogel of hyaluronic acid: synthesis, surface morphology, and solid state NMR. J Am Chem Soc 1994;116:7515. Pouyani T, Prestwich GD. Functionalized derivatives of hyaluronic acid oligosaccharides: drug carriers and novel biomaterials. Bioconjug Chem 1994;5:339–47. Goetz DJ, Greif DM, Ding H, Camphausen RT, Howes S, Comess KM, Snapp KR, Kansas GS, Luscinskas FW. Isolated Pselectin glycoprotein ligand-1 dynamic adhesion to P- and Eselectin. J Cell Biol 1997;137:509–19. Blackwell JE, Dagia NM, Dickerson JB, Berg EL, Goetz DJ. Ligand coated nanosphere adhesion to E- and P-selectin under static and flow conditions. Ann Biomed Eng 2001;29:523–33.

ARTICLE IN PRESS Y.H. Yun et al. / Biomaterials 25 (2004) 147–157 [24] Dickerson JB, Blackwell JE, Ou JJ, Patil VR, Goetz DJ. Limited adhesion of biodegradable microspheres to E- and P-selectin under flow. Biotechnol Bioeng 2001;73:500–9. [25] Kansas GS. Selectins and their ligands: current concepts and controversies. Blood 1996;88:3259–87. [26] Luscinskas FW, Gimbrone Jr MA. Endothelial-dependent mechanisms in chronic inflammatory leukocyte recruitment. Annu Rev Med 1996;47:413–21. [27] Fox SB, Turner GD, Gatter KC, Harris AL. The increased expression of adhesion molecules ICAM-3, E- and P-selectins on breast cancer endothelium. J Pathol 1995;177:369–76. [28] Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980;102:344–52. [29] Wang D, Robinson DR, Kwon GS, Samuel J. Encapsulation of plasmid DNA in biodegradable poly(d,l-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. J Control Release 1999;57:9–18. [30] Crutchfield KL, Shinde Patil VR, Campbell CJ, Parkos CA, Allport JR, Goetz DJ. CD11b/CD18-coated microspheres attach to E-selectin under flow. J Leukoc Biol 2000;67: 196–205. [31] Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone Jr MA. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci USA 1987;84: 9238–42. [32] Sako D, Chang XJ, Barone KM, Vachino G, White HM, Shaw G, Veldman GM, Bean KM, Ahern TJ, Furie B, et al. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell 1993;75:1179–86. [33] He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol 1998;160:1029–35. [34] Gopalan PK, Jones DA, McIntire LV, Smith CW. Cell adhesion under hydrodynamic flow conditions. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current protocols in immunology. New York, NY: Wiley; 1996. p. 7.29.1– 7.29.23. [35] Shinde Patil VR, Campbell CJ, Yun YH, Slack SM, Goetz DJ. Particle diameter influences adhesion under flow. Biophys J 2001;80:1733–43. [36] Jones DH, Corris S, McDonald S, Clegg JC, Farrar GH. Poly(dllactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 1997;15:814–7. [37] Hsu YY, Hao T, Hedley ML. Comparison of process parameters for microencapsulation of plasmid DNA in poly(d,l-lactic-coglycolic) acid microspheres. J Drug Target 1999;7:313–23. [38] Tinsley-Bown AM, Fretwell R, Dowsett AB, Davis SL, Farrar GH. Formulation of poly(d,l-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. J Control Release 2000;66:229–41.

157

[39] Benedetti LM, Topp EM, Stella VJ. Microspheres of hyaluronic acid esters—fabrications methods and in vitro hydrocortisone release. J Control Release 1990;13:33–41. [40] Lim ST, Martin GP, Berry DJ, Brown MB. Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. J Control Release 2000;66:281–92. [41] Deasy PB. Microencapsulation and related drug processes. New York, NY: Marcel Dekker, Inc.; 1984. p. 61–95. [42] Carmeliet P, Conway EM. Growing better blood vessels. Nat Biotechnol 2001;19:1019–20. [43] Baek S, March KL. Gene therapy for restenosis: getting nearer the heart of the matter. Circ Res 1998;82:295–305. [44] Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric system for dual growth factor delivery. Nat Biotechnol 2001;19:1029–34. [45] Cybulsky MI, Gimbrone Jr MA. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 1991;251:788–91. [46] Fleming S, Jones DB. Antigenic heterogeneity of renal endothelium. J Pathol 1989;158:319–23. [47] Ponder BA, Wilkinson MM. Organ-related differences in binding of Dolichos biflorus agglutinin to vascular endothelium. Dev Biol 1983;96:535–41. [48] Spragg DD, Alford DR, Greferath R, Larsen CE, Lee KD, Gurtner GC, Cybulsky MI, Tosi PF, Nicolau C, Gimbrone Jr MA. Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system. Proc Natl Acad Sci USA 1997;94: 8795–800. [49] Bendas G, Krause A, Schmidt R, Vogel J, Rothe U. Selectins as new targets for immunoliposome-mediated drug delivery. A potential way of anti-inflammatory therapy. Pharm Acta Helv 1998;73:19–26. [50] Bloemen PG, Henricks PA, van Bloois L, van den Tweel MC, Bloem AC, Nijkamp FP, Crommelin DJ, Storm G. Adhesion molecules: a new target for immunoliposome-mediated drug delivery. FEBS Lett 1995;357:140–4. [51] Everts M, Kok RJ, Asgeirsdottir SA, Melgert BN, Moolenaar TJ, Koning GA, van Luyn MJ, Meijer DK, Molema G. Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate. J Immunol 2002;168:883–9. [52] Wiewrodt R, Thomas AP, Cipelletti L, Christofidou-Solomidou M, Weitz DA, Feinstein SI, Schaffer D, Albelda SM, Koval M, Muzykantov VR. Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells. Blood 2002;99:912–22. [53] Dagia NM, Goetz DJ. A Proteasome inhibitor reduces concurrent, sequential, and long term IL-1b and TNF-a induced endothelial cell adhesion molecule expression and resultant adhesion. Am J Physiol Cell Physiol 2003; 10.1152/ajpcell.00102.2003 (in press).