Coacervate microspheres as carriers of recombinant adenoviruses

Subramanian Kalyanasundaram,1 Sharon Feinstein,1 Jennifer P. Nicholson,2 Kam W. Leong,1 and Robert I. Garver, Jr.2 1Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205; and 2Division of Pulmonary and Critical Care Medicine, University of Alabama School of Medicine and Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 35294.

The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8–10 ␮M in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

Key words: Gene therapy; adenoviridae; microspheres.

ultiple vector systems have been proposed for rus (CMV)-directed transgene expression by a transcrip- Mcancer gene therapy, but arguably the best avail- tional mechanism.5 able is the E1-defective recombinant adenovirus (rAd). Both of the strategies described would be enhanced by Advantages include the extensive understanding of the methods for copackaging both the virus and the ampli- biology of the virus, the well-established methods for the fying agent into a single “vector.” In this context, the first generation of high-titer rAds, and the generally high step is the development of a system that packages Ad in expression of the viral transgene, among others (re- a bioactive and releasable form. It was hypothesized viewed in Ref. 1). Important limitations of the existing here that microspheres might be capable of fulfilling adenoviral vectors include the inability to achieve the these two important goals. Microspheres might not only following: (a) targeted delivery of the rAds by systemic offer opportunities for the codelivery of virus and am- administration; (b) extended, local delivery of the ade- plifying agents but may also provide a means of achiev- novirus (Ad) without the requirement for frequent ing extended virus release by modulation of the formu- redosing; and (c) transduction of a large fraction of cells lation. In addition, in vivo biodistribution of the comprising tumor masses. microspheres could be modified by changes in size or by With respect to the latter limitation, researchers at the attachment of targeting moieties to the microsphere this laboratory have been exploring means of amplifying exterior without requiring any modification of the en- conventional adenoviral transduction efficiency and capsulated virus. As a first step toward investigating the transgene expression. We have shown that codelivery of possible utility of microspheres for addressing the short- an E1-defective Ad and either plasmid or RNA encod- comings of the present Ad vector systems, coacervate ing the deleted E1 functions amplifies adenoviral trans- microspheres were used to encapsulate rAd in the gene expression and the “therapeutic effect.”2–4 In a experiments reported here. different approach, we have also shown that the phar- macologic modulation of rAd-infected cells by histone MATERIALS AND METHODS deacetylase inhibitors markedly amplified cytomegalovi- Microsphere synthesis Microspheres were created by the coacervation of gelatin and Received March 4, 1998; Accepted May 15, 1998. alginate in the presence of rAd. AdCMV-luc (108 plaque- Address correspondence and reprint requests to Dr. Robert I. Garver, forming units (PFU))6 was mixed with 1 mL of 0.1% sodium 701 South 19th Street, LHRB 339, Birmingham, AL 35294. alginate solution. A total of 1 mL of 3.5% gelatin solution was © 1999 Stockton Press 0929-1903/99/$12.00/ϩ0 heated to 50°C and added while gently vortexing the alginate-

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Figure 1. SEM (ϫ3960 magnification) of microspheres containing rAd. virus mixture. Under these conditions, a phase separation In vitro release assays occurred with the formation of microspheres containing en- Serial sampling of media incubated with the microspheres was trapped Ad. The microsphere solution was treated with CaCl2 of varying concentrations as indicated in the text (see Fig 3) for performed to assess the time-dependent release of rAd from varying amounts of stabilization. The microspheres were then the microspheres. By quantifying the virus remaining in the separated from the supernatant by sucrose gradient centrifu- supernatant, it was estimated that 72–75% of the rAd was gation. The microspheres were separated from the sucrose by encapsulated in the microspheres used for these experiments. dialyzing against a 1% mannitol solution for 4 hours and were By quantifying the gelatin remaining in the liquid phase after subsequently frozen, lyophilized, and stored at 4°C. coacervation, it was estimated that the total mass of the microspheres was 6.5 mg Ϯ 1%, which was subsequently Physical characterization divided into three equal aliquots for the calcium stabilization. One-third of each lot of the microspheres was hydrated in 3 Microspheres were rehydrated in a minimal volume of water, mL of media (Dulbecco’s modified Eagle’s medium/F12 sup- air dried, and examined by scanning electron microscopy plemented with 10% fetal calf serum, 2 mM glutamine, and (SEM) to characterize the external features of the micro- antibiotics). The microspheres were transferred to microfuge spheres. The AdCMV-luc virus was labeled by incubation with tubes and incubated at 37°C with agitation for the duration of a fluorescently tagged antibody (Ab) directed against a viral the assay. At each sampling time, as defined in the text (see Fig capsid protein. Fluorescein isothiocyanate (FITC)-labeled Ab 3), the tubes were centrifuged at 300 ϫ g at room temperature to the Ad hexon protein was commercially obtained (anti-Ad to pellet the microspheres and the supernatant was carefully type 2 hexon, Accurate Chemical and Scientific Corporation, aspirated and replaced with fresh media. Samples were stored Westbury, NY). Measured amounts of the Ab and the virus at Ϫ70°C. The rAd present in each sample was quantified by were mixed to achieve a 1:1 Ab to rAd stoichiometric ratio and plaque assay using our previously described methods.4 allowed to react at room temperature for 30 minutes. The unbound Ab was removed by dialysis against phosphate- In vivo release assays buffered saline overnight. Microspheres were prepared for microscopic examination under conditions similar to those The ability of the microspheres to release rAd was assessed by used for the SEM, irradiated by a 488-Å argon laser, and quantification of the adenoviral transgene expression in human light-sectioned by confocal microscopy. lung cancer xenografts that had received a single injection of

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Figure 2. Confocal microscopy of microspheres containing rAd. Ultraviolet-irradiated microspheres made with FITC-labeled Ad were examined in 1-␮M sections starting at the near surface of the sphere (a) and progressing to the far surface of the sphere (f). Labeled virus is bright yellow. the microspheres. Athymic nude mice were engrafted with the Release assays showed that bioactive virus was re- H1299 cell lines by a subcutaneous injection (2 ϫ 106 cells/ leased from the coacervate microspheres in a time- injection) into the flanks using our previously described meth- dependent fashion (Fig 3). Three lots of microspheres 3 ϳ ods. At the time that tumors were 10–15 mm in greatest were made with identical amounts of virus, alginate, and diameter, they were injected with microspheres or free gelatin, followed by different calcium cross-linking con- AdCMV-luc by a single intratumoral (i.t.) injection (60 ␮L volume) with a 27-gauge needle. The microspheres were ditions for stabilization. In these assays, all three lots of suspended at a concentration of 2 ␮g/␮L. The free Ad was spheres released significant amounts of virus at the diluted in media to achieve the required concentrations for initial 30-minute sampling point. Those spheres that delivery of 105 or 106 PFU. Mice were sacrificed at 3 days were stabilized with 3% and 1.5% calcium had similar postinjection, and the tumor nodules were removed by dissec- release curves that showed a sustained release of Ad tion. Each nodule was weighed and homogenized; the lucif- over the first 3.5 hours, although the majority of the virus erase activity was normalized to the protein concentration, and was released in the first 1.5 hours. In contrast, the 0.75% tumor mass was determined using our previously described 3 stabilized spheres released minimal additional amounts techniques. of virus beyond the 30-minute sampling point. Although the pattern of release in the 3.0% and 1.5% spheres was similar, the total amount of virus released varied signif- RESULTS AND DISCUSSION icantly between these two lots. The 3.0% spheres re- leased a total of 2.4 ϫ 106 PFU/mL over the entire assay The coacervation of gelatin and alginate in the presence period compared with the 1.5% lot, which released a of rAd yielded spheres containing the Ad. SEM showed total of 15.0 ϫ 106 PFU/mL. The 0.75% lot released a that the coacervate was composed of regular spheres total of 4.5 ϫ 106 PFU/mL. Based on these results, it that ranged in diameter from 0.8 to 10 ␮M (Fig 1). seems that the microspheres stabilized with the 1.5% Confocal microscopy of microspheres made with FITC- calcium provided a sustained release of the largest labeled Ad showed that the microspheres contained Ad amount of virus. in a relatively uniform distribution (Fig 2). Human lung cancer xenografts displayed luciferase

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Figure 3. Time-dependent release of Ad from coacervate microspheres. Three lots of microspheres were each made with the same amounts of virus, gelatin, and alginate; however, these lots varied in the calcium stabilization conditions that are indicated in the legend above the graph. activity after i.t. administration of the microspheres PFU/mL over 48 hours with a total assay volume of 3 containing the AdCMV-luc (Fig 4). The non-small-cell mL, to achieve a total of 4.5 ϫ 107 PFU. As explained in lung cancer cell line H1299 was engrafted into the flanks Materials and Methods, it was estimated that ϳ2.5 ϫ 107 of nude mice to form tumor nodules. Microspheres, or PFU were encapsulated in each aliquot of the spheres free rAds, were administered by a single i.t. injection of tested in the release assays. Based on these estimates, each nodule. The relative gene transfer accomplished by the microspheres released more bioactive virus than was the microspheres or by free rAd was estimated by incorporated into the spheres, which most likely reflects quantification of the luciferase activity. This analysis the combined degree of error in the estimates of virus showed that the nodules treated with the microspheres incorporation and in the plaque assay quantifications of had luciferase activities that were significantly greater virus used for both input and output determinations. than those treated with 105 PFU of free viruses (P Ͻ .04) Despite these limitations of accuracy, these results and insignificantly different from the nodules treated strongly suggest that the process of microsphere synthe- with 106 PFU of free virus (P Ͼ .56). sis and lyophilization did not significantly reduce the Two lines of evidence shown here demonstrate that viral bioactivity. The total amount of virus released was bioactive rAd could be encapsulated in, and subse- less in the two other microsphere formulations and was quently released from, coacervate microspheres com- most likely the consequence of retained virus, because posed of gelatin and alginate. First, the confocal micro- the calcium concentration was not expected to signifi- scopic characterization of the microspheres made with cantly modify the bioactivity. tagged rAd showed that virus was present in a relatively The choice of the coacervate microsphere formulation homogeneous distribution within the spheres. Second, in was based in part on the gentle conditions required for vitro release studies with the microspheres showed a microsphere formation before lyophilization. We ini- time-dependent release of virus. It is important to note tially explored microspheres made with alginate chelated that the microspheres were subjected to sucrose gradient with calcium, because those conditions were also favor- centrifugation and dialysis before lyophilization to avoid able for rAd bioactivity (data not shown). However, the the presence of free rAd in the final, lyophilized prepa- chelated alginate microspheres had several unfavorable rations. Furthermore, free virus would not explain the characteristics. First, they had to be stored in the aque- sustained release observed in the microsphere formula- ous phase, either liquid or frozen. In the frozen state, the tions tested here. virus bioactivity was variably affected because it was not The results of the in vitro assays suggested that the in a glycerol-containing buffer. In the liquid state, virus rAd tolerated these encapsulation conditions with a started diffusing from the microspheres immediately minimal loss of bioactivity. In the microspheres stabi- after formation, so that the characteristics of each lot of lized with 1.5% calcium, the virus released was 1.5 ϫ 107 spheres changed continually over time. Second, the

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Figure 4. In vivo release of Ad from coacervate microspheres. The luciferase activity in tumor nodules after the administration of microspheres containing AdCMV-luc (mcsp), 105 free AdCMV-luc (free 1eϩ05), or 106 free AdCMV-luc (free 1eϩ06) is shown. For microspheres, n ϭ 12; for the other two groups, n ϭ 6 for each. Luciferase activity is shown on the ordinate as relative light units (RLU) normalized to the protein content of the lysate Ϯ SEM. formulations required to permit virus release resulted in assessment of later timepoints after transduction may delicate spheres that did not store or ship well. have yielded different results. We were originally dissuaded from pursuing the coac- We have reported previously that similar coacervate ervate microspheres because of the lyophilization re- microspheres could be conjugated at the surface with quirement. In preliminary experiments, it was found that monoclonal Abs that preferentially direct the micro- lyophilization of free Ad resulted in a major loss of Ad spheres to the lung or to the hematopoietic system.8 It bioactivity. However, it was unexpectedly found that seems reasonable to speculate that similar strategies lyophilization of the virus within the microsphere greatly could be used with the Ad-containing microspheres as a minimized the bioactivity loss. The ability to store the means of modifying biodistribution in vivo. microspheres at 4°C in the lyophilized state was a major advantage in that the characteristics of each lot of spheres changed more slowly over time than was the ACKNOWLEDGMENTS case with alginate spheres; fragility was no longer a problem. We thank R. Gerard for providing the AdCMV-luc virus used The importance of the sustained release of a vector at for this study. This study was supported in part by a grant to R.I.G. and K.W.L. from the American Heart Association. the target site is unclear. Earlier findings by Culver et al, in which administration of retroviral producer cells into a tumor mass resulted in a higher transduction efficiency REFERENCES than did bolus i.t. administration, suggested that sus- 7 tained vector release was important. Clearly, the supe- 1. Curiel DT, Garver RI, Jr. Adenoviral vectors for gene rior transduction efficiency with the retroviral producer therapy of inherited and acquired disorders of the lung. In: cells may also have been in part the consequence of the Brigham KL, ed. Gene Therapy for Diseases of the Lung. greater virus dose provided by the in situ viral producer New York: Marcel Dekker; 1997:29–52. cells compared with bolus administration. In the findings 2. Goldsmith KT, Curiel DT, Engler JA, et al. Trans-comple- presented here, the amount of microspheres adminis- mentation of an E1A-deleted adenovirus with codelivered tered was estimated to have 1.4 ϫ 106 PFU, which E1A sequences to make recombinant adenoviral producer cells. Hum Gene Ther. 1994;5:1341–1348. resulted in an estimated gene transfer efficiency that was 6 3. Dion LD, Goldsmith KT, Garver RI, Jr. Quantitative and comparable with the tumors treated with 10 PFU alone. in vivo activity of adenoviral-producing cells made by Although these results did not demonstrate a superior cotransduction of a replication-defective adenovirus and a gene transfer efficiency under these conditions, it is replication-enabling plasmid. Cancer Gene Ther. 1996;3: possible that differently formulated microspheres or an 230–237.

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