Folic Acid-Conjugated Protein Cages of a Plant Virus - American

Bioconjugate Chem. 2007, 18, 836−843

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Folic Acid-Conjugated Protein Cages of a Plant Virus: A Novel Delivery Platform for Doxorubicin Yupeng Ren,† Sek Man Wong,‡,§ and Lee-Yong Lim†,|,* Department of Pharmacy and Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543, Temasek Life Sciences Laboratory, 1 Research Link, Singapore, 117604, and Pharmacy, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA 6009, Australia. Received November 20, 2006; Revised Manuscript Received January 22, 2007

The protein cage of a plant virus may provide a template for monodispersed nanosized systems for drug delivery. Using the Hibiscus chlorotic ringspot virus (HCRSV) as a model plant virus, we have prepared nanosized protein cages (30 nm) capable of encapsulating the anticancer drug, doxorubicin. The technique utilized the simultaneous encapsulation of a polyprotic acid of mw 200 kDa to produce an encapsulation efficiency for doxorubicin of about 7.5%. Folic acid was conjugated onto the capsids to impart cancer-targeting capability. The resultant nanosized systems improved the uptake and cytotoxicity of doxorubicin in the ovarian cancer cells, OVCAR-3, with statistical significance. Plant virus capsids may therefore provide viable templates for targeted drug delivery in cancer chemotherapy.

INTRODUCTION Plant viruses which have genomic materials encapsidated within protein cages may provide viable delivery platforms for drugs (1). Unlike viruses isolated from animal origins, plant viruses are less pathogenic (2, 3) and may avoid the immunogenicity response in animals (4). The protein cages, or capsids, offer versatility for drug loading. Compounds can either be physically encapsulated within the capsids following the removal of native RNA, or chemically conjugated onto the capsid. As the capsids are assembled from identical coat protein units, they are exact in molecular weight (mw), uniform in size, and precise in structure (5). This homogeneity in size and shape is an advantage over most nanoscale drug delivery systems prepared from random aggregation of macromolecules such as lipids or polymers (6). Plant viral capsids provide other advantages for drug delivery. Amino acids in the coat protein are potential chemical conjugation sites for ligand attachment to develop stealth and/or targeting drug delivery platforms. Both macromolecules, such as polyethylene glycol or entire proteins (4, 7), and small inorganic atoms, such as gold (8), have been conjugated onto the surface of viral protein cages to yield novel properties. The insertion of foreign peptides by recombinant DNA technology can further entitle the capsids to specific bioactivity (9, 10). Of the wide variety of plant viruses known to man, the icosahedral plant viruses are to date the most researched as vessels for foreign material loading. By adjusting the environmental pH and ion content, the capsids of these viruses can be transformed from assembled to swollen to disassembled states (11). This permits the removal of native viral RNA from the coat proteins, which are then purified and reassembled into protein cages for cargo loading. Proof of concept was first * Corresponding author. E-mail: [email protected]; telephone: 61-8-64884413; fax: 61-8-64881025. † Department of Pharmacy, National University of Singapore. ‡ Department of Biological Sciences, National University of Singapore. § Temasek Life Sciences Laboratory. | Pharmacy, School of Biomedical, Bimolecular and Chemical Sciences, University of Western Australia.

demonstrated with the Cowpea chlorotic mottle virus (CCMV)1 (12, 13). The protein cages of CCMV could host foreign ions as well as polyacids (12), the capacity to host different ions being made possible by modulating the inner surface charges of the protein cage through gene mutation (13). More recently, gold nanoparticles of appropriate size were also encapsidated within the CCMV-derived protein cages (14, 15). Success in these experiments has prompted an interest in using the plant viral protein cages as platforms for drug delivery. Loading of cargo has been achieved through chemical conjugation of the molecules with specific amino acid groups presenting on the interior surface of the viral protein cages (16, 17). In another development, the anticancer drug, doxorubicin, was covalently attached to the interior surface of small heat-shocked viral protein cages (18). To date, there is no report on the loading of small chemotherapeutic drugs into plant viral protein cages by physical encapsulation, although this method has proven effective for the loading of crystals (12, 13), polymers (12), and gold nanoparticles (14, 15). Our previous research on another plant virus, the HCRSV, has established two prerequisites for the successful loading and retention of exogenous material in the viral protein cages (19). First, the material should possess a negative charge so that it will bind with the coat protein and initiate the reassembly of the icosahedral capsid structure. Second, the material should have an adequately high mw to 1 Abbreviations: HCRSV, Hibiscus chlorotic ringspot virus; CCMV, Cowpea chlorotic mottle virus; PSA, polystyrenesulfonic acid; PCDox, doxorubicin-loaded capsids; fPC-Dox, folic acid-conjugated, doxorubicin-loaded capsids; FR, folic acid receptor; EDTA, ethylenediamine-N,N,N′,N′-tetraacetic acid; DTT, dithiothreitol; PMSF, phenylmethanesulfonyl fluoride; EDAC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; TEM, transmission electron microscope; Dox-PSA, doxorubicin complexed with PSA; Dox-CP, doxorubicin incubated with empty viral capsids; PSA-PC, preformed PSA-loaded capsids; LE, loading efficiency, EE, encapsulation efficiency; RE, reassembly efficiency; N, the number of doxorubicin molecules encapsidated within each capsid; fPC-Dox + f, fPC-Dox in the presence of 1 mM of folic acid; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density.

10.1021/bc060361p CCC: $37.00 © 2007 American Chemical Society Published on Web 04/04/2007

Drug Loading by Polyacid Association in Viral Capsids

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cells (22, 23). In this study, folic acid was conjugated on the protein cages to promote the cellular uptake of drug by cancer cells.

EXPERIMENTAL PROCEDURES

Figure 1. Schematic illustration of the preparation of doxorubicinloaded viral protein cage without (PC-Dox) and with folic acid conjugation (fPC-Dox). Steps A1 and B2 are indicative of the removal of viral RNA from the plant virus and purification of coat proteins. Steps A2 and B3 involve the encapsulation of polyacid and doxorubicin during the reassembly of protein cage. Step B1 refers to the conjugation of folic acid onto the viral protein coat.

avoid excessive leaching of encapsidated material through the cavities present on the capsid surface upon dilution. Doxorubicin, like many other anticancer drugs, is a small molecule (mw ) 545 Da), and it is positively charged (pKa ) 8.4) in physiological fluids as well as under the conditions for reassembling of plant virus capsids (20). These properties made it difficult to load and retain doxorubicin within the plant virus capsid by simple encapsulation during the reassembly of the protein cage. To impart the requisite size and charge to the anticancer agent, we employed the aid of a polyacid. This method, which we termed “polyacid association”, involved the simultaneous loading of doxorubicin with polystyrenesulfonic acid (PSA) of mw 200 kDa into the capsid of the HCRSV. The 3:1 (PSA:drug) w/w ratio of mixing ensures the attraction of doxorubicin molecules to the polyacid by electrostatic forces, and a net negative charge for the resultant complex. The complex thus provided the nucleus for binding with, and initiation of, the final reassembly of coat proteins into the viral capsids (Figure 1). The resultant system was denoted as PC-Dox. We also prepared folic acid-conjugated equivalents, denoted as fPC-Dox, where the HCRSV viruses were conjugated with folic acid before they were disassembled for doxorubicin-PSA encapsulation. Folic acid has been widely studied as a small molecule-ligand for targeting drug delivery systems to cancer cells. The principle of this application is based on the differential expression of folic acid receptor (FR) on normal and cancer cells. FR expression is elevated in malignant cancer cells, e.g., cancer cells of ovary, uterus, and mesothelium (21, 22), while most normal cells express low levels of FR. Proof of concept of the folic acid-mediated targeting systems have been shown with the delivery of drugs and liposomes to a variety of cancer

Materials. HCRSV was purified from infected kenaf leaves (24). RPMI 1640 medium and folic acid-deficient RPMI 1640 medium were purchased from Sigma Chemical Co. (Steinheim, Germany) and supplemented with 10% fetal bovine serum (Gibco BRL Life Technologies, Grand Island, NY). OVCAR-3 cell line (Passage 22 to 32) was obtained from ATCC (Manassas, VA) and maintained in folic acid-deficient RPMI 1640 medium for 2 months before use (25). CCL-186 cell line (Passage 15 to 25) was obtained from ATCC and cultured in RPMI 1640 medium. Polystyrenesulfonic acid and folic acid were from Sigma (St. Louis, MO). Doxorubicin was purchased from Pharmacia (Bentley, WA, Australia). Preparation of Doxorubicin-Loaded Capsids (PC-Dox). The coat protein of HCRSV was purified by dialysis against 1000 mL of pH 8.0 Tris buffer (50 mM Tris/base, 5 mM EDTA, 2 mM DTT, 0.2 mM PMSF, pH 8.0) for 12 h followed by centrifugation to remove viral RNA as described previously (19). After further dialysis against 1000 mL of pH 7.0 Tris buffer (50 mM Tris/base, 5 mM EDTA, 2 mM DTT, 0.2 mM PMSF, 1 M NaCl, pH 7.0) for 4 h, 1 mL of the coat protein solution (1 mg/mL) was incubated with 0.5 mL of doxorubicin-PSA solution (0.2 mg/mL of doxorubicin, 0.6 mg/mL of PSA (mw ) 200 kDa) in pH 7.0 Tris buffer) for 8 h at 4 °C. The solution was adjusted to pH 5 with sodium acetate buffer (1 M sodium acetate, pH 5.0), and CaCl2 was added to a final concentration of 5 mM to reassemble the protein cages. The solution was centrifuged under 15 000g for 10 min, and the supernatant was put on a 15% sucrose cushion and ultracentrifuged at 100 000g (SW41 rotor) for 1 h to sediment the protein cages. The pelleted PC-Dox was suspended in 0.1 mL of resuspension buffer (50 mM sodium acetate, 20 mM NaCl, 5 mM EDTA, 20 mM CaCl2, pH 5.0) and stored at 4 °C until use. Preparation of Folic Acid-Conjugated, DoxorubicinLoaded Capsids (fPC-Dox). The carbodiimide coupling method was used to conjugate folic acid onto the capsids (2630). Five milligrams of folic acid, 50 mg of 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDAC), and 50 mg of N-hydroxysuccinimide (NHS) were dissolved in 50 mL of bicarbonate buffer (50 mM NaHCO3, pH 6) at ambient temperature. Five milligrams of HCRSV was then incubated with the reaction mix at 4 °C for 8 h. The solution was concentrated to about 0.5 mL by ultrafiltration and washed thrice with 10 mL of resuspension buffer to remove the unreacted materials. Dialysis against Tris buffers as described above was employed to remove the viral RNA and purify the coat protein. To determine the conjugation efficiency of a sample, the conjugated folic acid was quantified based on OD360nm using an extinction coefficient of 5312 mol-1 cm-1 (Beckman Du 640B spectrometer, CA) (28-32), while the protein content was determined by the bis-cinchoninic acid assay (TECAN SpectraFluor, MA) (33). Reassembly of the purified folic acidconjugated coat protein into protein cages, with simultaneous loading of doxorubicin and polystyrenesulfonic acid, were also conducted using methods similar to those described for the unconjugated protein coats. Characterization of PC-Dox and fPC-Dox. The morphology of HCRSV, PC-Dox, and fPC-Dox were observed under a transmission electron microscope (TEM) (CM10, Philips Electronic Instruments Co, NJ) after staining with 1% of phosphotungstic acid. Sucrose gradient centrifugation was performed to confirm the existence of the drug-loaded protein cages. About 0.5 mL of PC-Dox and fPC-Dox in resuspension

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buffer was centrifuged on 10-40% sucrose gradient at 100 000g for 3 h. Fractions of the sucrose gradient were collected for OD485nm measurement to detect for doxorubicin. Control samples, including free doxorubicin, doxorubicin complexed with PSA at a weight ratio of 3:1 (Dox-PSA), doxorubicin incubated with empty viral capsids under reassembly conditions (Dox-CP), and doxorubicin incubated with preformed PSAloaded capsids (PSA-PC) (19), were also subjected to the sucrose gradient centrifugation. Native electrophoresis of PC-Dox and fPC-Dox was performed on 1% agarose gel under 5 V/cm for 1 h (Bio-Rad Sub-Cell electrophoresis system). Bands of coat protein were visualized by Coomassie blue staining, while the doxorubicin bands were visualized under ultraviolet light. Loading efficiency (LE), encapsulation efficiency (EE), reassembly efficiency (RE), and the number of doxorubicin molecules encapsidated within each capsid (N) were calculated using eqs 1-4. The amount of doxorubicin was calculated by comparing the OD485 nm of sample and standard solutions, both of which were diluted with resuspension buffer (pH 5). The amount of reassembled coat protein (protein cage) was quantified by the BCA assay after the encapsidated doxorubicin was removed via a 6-day dialysis against PBS (pH 7.4). Drug release experiments were performed by the dialysis method (34). Fifty microliters of PC-Dox and fPC-Dox suspensions, with an equivalent doxorubicin concentration of 100 µg/mL, were placed in separate dialysis tubes (mw cutoff 12 400, Sigma-Aldrich) and dialyzed against 25 mL of PBS (pH 7.4) at 37 °C. At specified time points, 0.2 mL of the dialysis buffer was sampled for fluorescence measurements (Ex 480 nm, Em 535 nm) to quantify the released doxorubicin.

LE (%) ) weight doxorubicin loaded/weight protein cage × 100%

(1)

EE ) weight doxorubicin loaded/weight total doxorubicin used × 100% (2) RE ) weight protein cage/weight total coat protein used × 100% N ) LE/mw doxorubicin × (mw coat protein × 180)

(3) (4)

The mwdoxorubicin and mwcoat protein values were 545 and 37 000, respectively. Cellular Uptake of Doxorubicin. OVCAR-3 and CCL-186 cells were subcultured in six-well plates at concentrations of 100 000 cells/well. After 24 h, the cells were incubated with doxorubicin, PC-Dox, and fPC-Dox at equivalent doxorubicin concentration of 5 µg/mL. In the case of fPC-Dox, the incubation was also conducted in the presence of 1 mM of folic acid (fPC-Dox + f). The cells were washed thrice in ice-cold PBS to remove the samples, then lyzed with 0.5 mL of lysis buffer (50 mM Tris, 0.8% Triton, 0.2% SDS, pH 7.4). Cellassociated doxorubicin was determined by measuring the fluorescence of the lysate using a microplate reader (TECAN SpectraFluor, MA, Ex 485 nm and Em 535 nm) (35). Confocal Microscopy. Cells were cultured in Lab-Tek chambered glass system and incubated for 1 h with samples of doxorubicin, PC-Dox, fPC-Dox and fPC-Dox + f, at a doxorubicin concentration of 5 µg/mL. The cells were washed thrice with PBS and the intracellular doxorubicin was visualized under a confocal microscope (CSLM, Zeiss Axiovert 200M, Oberkochen, Germany, Ex480nm and Em540nm) (36). MTT Assay. PC-Dox, fPC-Dox and fPC-Dox + f samples were diluted with folic acid-deficient RPMI 1640 medium to give doxorubicin concentration in the range of 0.005 to 30 µg/ mL. OVCAR-3 and CCL-186 cells were subcultured in 96-well plates at a density of 20 000 and 5000 cells/well, respectively. After 24 h, the culture medium was aspirated, and the cells were

Figure 2. Optical density of fractions obtained from sucrose gradients. HCRSV was detected at 260 nm while other samples were detected at 485 nm. (a) Doxorubicin in control samples stayed at the top of the sucrose gradient after centrifugation. (b) Doxorubicin in PC-Dox and fPC-Dox showed greater propensity to sediment after centrifugation.

incubated with 0.1 mL of doxorubicin, PC-Dox, fPC-Dox, or fPC-Dox + f samples for 2 h. The cells were then washed thrice in ice-cold PBS and incubated with 0.2 mL of fresh cell culture medium for 3 days. Cell viability was determined by the MTT assay, and IC50 values were calculated using the nonlinear regression sigmoidal dose-response equation (Prism Version 3.00) (25, 37-39).

RESULTS AND DISCUSSION Fractions isolated from the sucrose gradient centrifugation and subjected to optical density (OD) measurements indicated that the free doxorubicin, Dox-PSA, Dox-CP, and doxorubicin incubated with preformed PSA-PC were retained at the top (Figure 2, a), while native HCRSV were retained in fraction 13 of the sucrose gradient. In contrast, sucrose gradient centrifugation analysis of PC-Dox and fPC-Dox showed high doxorubicin content in fractions 11-15, with peak doxorubicin content noted in fraction 12 (Figure 2, b). The sedimentation rate of doxorubicin in these samples is higher than that of free doxorubicin, indicating that the drug had been loaded into the viral capsids. Interestingly, the sedimentation rates of PC-Dox and fPC-Dox were comparable with that of native HCRSV, suggesting similar cargo loads in the viral capsids of these samples. It may therefore be concluded that successful drug encapsulation was achieved in the PC-Dox and fPC-Dox samples but not in the Dox-CP and preformed PSA-PC samples. The failure to load doxorubicin into the preformed PSA-PC suggests that the drug could only be encapsulated during protein cage reassembly. The implication is that doxorubicin loaded in the PC-Dox and fPC-Dox samples was not likely to result from physical or chemical binding onto the surface of the protein cages but was contained within the HCRSV capsids. Native agarose gel electrophoresis was applied to analyze the electrostatic properties of the samples. As shown in Figure 3 a,


Figure 3. Native agarose gel electrophoresis. (a) Lanes 1-5 contain HCRSV, fPC-Dox, PC-Dox, doxorubicin, and doxorubicin-PSA complex. (b) Lanes 1-3 contain doxorubicin incubated with HCRSV, preformed PSA-PC, and PSA-fPC. The photos were taken by a digital camera, under ultraviolet light and after staining with coomassie blue, respectively.

free doxorubicin had migrated to the cathode, while PC-Dox, fPC-Dox, and the doxorubicin-PSA complex had moved in the opposite direction toward the anode. The relative positions of the bands indicate the opposing polarity of the samples and confirmed that the PSA-doxorubicin complex possessed a strong net negative charge. A comparison of the gels following coomassie blue staining showed that the doxorubicin band colocalized with the protein bands in the PC-Dox and fPCDox samples, once more underscoring the successful encapsulation of the drug in the viral capsids. Compared with the native HCRSV, the PC-Dox and fPC-Dox possessed a slightly lower charge density. In our previous study, HCRSV capsids loaded with PSA alone were determined to have the same negative charge density as the native HCRSV (19). Therefore, the lower charge density of PC-Dox and fPC-Dox could be attributed to the loaded doxorubicin. Samples in which doxorubicin was incubated with the preformed polyacid-loaded protein cages showed an absence of drug-loaded capsids (Figure 3, b). This negative control, together with that the results obtained from the sucrose gradient analysis, further confirms the encapsulation of doxorubicin during the process of capsid reassembly. Both PC-Dox and fPC-Dox appeared as spherical particles with a diameter of about 30 nm and were indistinguishable from the native HCRSV when viewed under the TEM (Figure 4). The “polyacid association” method was found to be efficient for capsid reassembly and drug loading. As shown in Table 1, doxorubicin loaded into PC-Dox and fPC-Dox amounted to 59 and 49%, respectively, of the initial doxorubicin load used to prepare the samples, while more than 60% of the coat protein was found to have reassembled into capsids in these two samples. Measured against the coat protein content, the drug loading efficiency (LE) was 7.5%, amounting to about 900 doxorubicin molecules encapsulated in each protein cage. The


encapsulated doxorubicin concentration would be in the vicinity of 300 mM, given that the inner diameter of the HCRSV protein cage was about 110 Å (40). Folic acid conjugation efficiency (weightfolic acid/weightcoat protein) for fPC-Dox was 1.9% (SD ) 0.1%, n ) 3), which translated to about two folic acid molecules conjugated to each coat protein. The folic acid is postulated to bind with the Lys amino acids on the exposed protruding domain of the coat proteins. Since the icosahedral HCRSV capsid is made up of 180 coat protein units (24, 40), the implication is that each fPCDox particle would have 360 conjugated folic acid molecules. The release of doxorubicin from PC-Dox and fPC-Dox following dilution was evident during sucrose gradient centrifugation and agarose gel electrophoresis. Fractions obtained from the sucrose gradient showed increasing doxorubicin concentration from fractions 0 to 10. Since the viral capsids were retained in fraction 12, this suggests that doxorubicin was released from the capsids as they sediment along the centrifugation path. Observation of the agarose gel also showed a strong trail of doxorubicin associated with the bands for PC-Dox and fPC-Dox (Figure 3, a). As the protein bands of PC-Dox and fPC-Dox (Figure 3, a) did not exhibit degradation trails, the protein cages were probably stable under the experimental conditions. The orange color trails and fluorescence trails might therefore be attributed to the doxorubicin which had been released from the capsids and had migrated toward the cathode. In Vitro drug release experiments were conducted under simulated physiological conditions (pH 7.4, 37 °C) (34). Sustained release was evident when the drug release profiles of PC-Dox and fPC-Dox were compared with that of free doxorubicin (Figure 5). More than 80% of the drug load was released from the free doxorubicin into the receptor chamber after 5 h, whereas the drug-loaded capsids yielded the same amount of doxorubicin only after 24 h. Drug release from the PC-Dox and fPC-Dox did not, however, occur at a constant rate. There was a fairly rapid release of doxorubicin in the initial phase, where 40% of the drug load was released in 4 h, followed by a slower pace of release of the remaining drug load from the capsids. The mechanisms that effect drug release upon dilution were probably similar to those that allowed doxorubicin to be loaded into the viral capsids in the first place, i.e., reversible electrostatic attraction with the polyacid and physical entrapment by the viral protein cage. The sustained-release profile, coupled with the almost complete release of the entire drug load, rendered the HCRSV protein cages as attractive platforms for controlled release drug delivery. However, to reduce drug release during storage, the samples should be stored in small volumes of resuspension buffer at low temperature (4 °C). In Vitro cellular uptake and cytotoxicity experiments were conducted on the ovarian epithelial adenocarcinoma OVCAR-3 cell line, and the CCL-186 cells, a human diploid fibroblast. The OVCAR-3 cells have been used to evaluate folic acidtargeting drug delivery systems because of the presence of overexpressed folic acid receptors in these cells (41-43). The CCL-186, on the other hand, served as a representative human normal cell and was used as a control to evaluate the capacity of the delivery systems to selectively deliver doxorubicin to cancer cells. Cellular uptake of doxorubicin was visualized by confocal microscopy and quantified by fluorimetry. Quantitative data showed a 2-fold higher cellular uptake of doxorubicin from fPC-Dox than from PC-Dox by the OVCAR-3 cells (Figure 6). Uptake of doxorubicin from PC-Dox was in fact no different from that of free doxorubicin, suggesting that the encapsulation of doxorubicin within the HCRSV capsids could not by itself aid in the intracellular accumulation of the drug in the

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Figure 4. TEM micrographs of (a) PC-Dox, (b) fPC-Dox, and (c) native HCRSV without modifications. The two drug-loaded viral protein cage particles were comparable in morphology and size with the native HCRSV. Table 1. PC-Dox and fPC-Dox Samples: Efficiency of Doxorubicin Loading and Reassembly of Coat Proteins into Capsids. Data Represent Mean ( SD, n ) 3. samples

EE (%)a

RE (%)b

LE (%)c

Nd

PC-Dox fPC-Dox

59.3 ( 13.7 48.7 ( 4.7

74.7 ( 12.1 63.8 ( 5.1

7.8 ( 0.5 7.5 ( 0.3

953 916

a

weightdoxorubicin/weightcoat protein expressed as a percentage. b weightdoxopercentage. percentage. capsid.

rubicin loaded/weightdoxorubicin used for preparation expressed as a c weight capsids/weightcoat protein used for preparation expressed as a d Number of doxorubicin molecules encapsidated within each

Figure 5. Doxorubicin release profile of PC-Dox, fPC-Dox, and free doxorubicin. More than 80% of free doxorubicin was released into the receptor chamber after 5 h whereas the drug-loaded capsids yielded the same amount of doxorubicin after 24 h.

OVCAR-3 cells. Coincubation of fPC-Dox with folic acid negated the higher cellular uptake of doxorubicin from this sample, implicating a folic acid-mediated uptake mechanism in the OVCAR-3 cells. Enhanced uptake of doxorubicin from fPC-Dox was not observed in the CCL-186 cells, which showed comparable drug uptake from the free doxorubicin, PCDox, and fPC-Dox samples. Confocal micrographs provided supporting evidence for the folic acid-mediated preferential uptake of fPC-Dox by the OVCAR-3 cells (Figure 7). OVCAR-3 cells incubated with fPC-Dox showed more intense fluorescence compared to cells incubated with free doxorubicin or PC-Dox. Upon the addition of folic acid to the fPC-Dox sample, however, the fluorescence exhibited by the cells was reduced to a level similar to those observed in cells incubated with PC-Dox or free doxorubicin. Doxorubicin typically accumulates in the nuclei of cells. However, for the fPC-Dox, which increased the intracellular fluorescence of doxorubicin, the fluorescence was not concentrated in the nuclei but in many bright small spots in the cytoplasm (Figure 7, c). The phenomenon was consistent with a FR-mediated uptake, whereby the drug-loaded capsids following endocytosis would be directed to the endosomes (44).

Figure 6. Cellular uptake of doxorubicin by (a) OVCAR-3 and (b) CCL-186 cells incubated with free doxorubicin, PC-Dox, and fPCDox. Uptake of fPC-Dox was also undertaken in the presence of folic acid (fPC-Dox + f). Data represent mean ( SD (n ) 3).

For the CCL-186 cells, slightly stronger fluorescence was observed in cells incubated with free doxorubicin than in cells incubated with PC-Dox or fPC-Dox. These observations are therefore in good agreement with the quantitative uptake data. Cytotoxicity of the various doxorubicin formulations against the OVCAR-3 and CCL-186 cells was assessed via the MTT assay after 2 h of exposure. The log C vs viability curves of OVCAR-3 cells showed that the fPC-Dox had higher cytotoxicity against this cell line compared with the other formulations (Figure 8). The PC-Dox formulation did not decrease the IC50doxorubicin, again affirming that the encapsulation of the drug alone did not confer additional benefits to its delivery to cancer cells. The IC50doxorubicin of fPC-Dox formulation (0.11 µg/mL) was observed to decrease 4-fold compared with the IC50doxorubicin of free doxorubicin (0.48 µg/mL). This enhancement of kill was again mediated by the folic acid conjugation on the capsid surface, for the addition of free folic acid



Figure 7. Confocal micrographs of OVCAR-3 cells incubated for 1 h with free doxorubicin (a), PC-Dox (b), fPC-Dox (c), and fPC-Dox in the presence of folic acid (d), and of CCL-186 cells incubated for 1 h with free doxorubicin (e), PC-Dox (f), fPC-Dox (g), and fPC-Dox in the presence of folic acid (h).

Figure 8. Viability of cells after treated by different formulations. (a) Cell viability of OVCAR-3. (b) Cell viability of CCL-186. The fPCDox was more efficient in inhibiting the growth of OVCAR-3 cells than other formulations while there was no such difference between the formulations in CCL-186 cells.

effectively abolished the added effect. A comparison of the IC50doxorubicin across cell types indicates that the CCL-186 cells, with the IC50doxorubicin of 1.95 µg/mL, were about 4-fold more resistant to the cytotoxicity of the drug than the OVCAR-3 cells. Interestingly, the encapsulation of doxorubicin in either the PCDox or fPC-Dox led to increased IC50doxorubicin of 3.4 µg/mL and 3.2 µg/mL against the CCL-186 cells, alluding to a conferred protection of the cells against the drug. The selective higher uptake rate and cytotoxicity of fPCDox in cancer cells relative to normal cells may be explained

by the up-regulation of folic acid receptors, in some cases approaching 2 orders of magnitude, in many cancer cell types (22, 45). To confirm a folic acid-mediated mechanism, the uptake and cytotoxicity experiments for fPC-Dox were conducted in the presence of high concentration of free folic acid, which was found to negate the benefits of fPC-Dox. This suggested the uptake of fPC-Dox by folic acid-mediated endocytosis in the OVCAR-3 cells. The preferential uptake of fPC-Dox led to its stronger cytotoxic effect, as denoted by the lower IC50doxorubicin value, in cancer cells. On the other hand, the uptake and cytotoxicity data suggest that the PC-Dox was not an efficient delivery system for cancer chemotherapy. This is reasonable considering the current lack of evidence to support cytoinvasive and cytotoxicity properties of plant viruses in animal cells. PC-Dox could, however, be useful in clinical applications where sustained drug release and/ or protection against cytotoxic drugs are desired. To conclude, a new “polyacid association” method was established to physically encapsulate the anticancer drug, doxorubicin, inside the protein cage of HCRSV at high loading efficiency. The conjugation of folic acid allows the viral capsids to selectively increase the uptake and cytotoxicity of doxorubicin in the ovarian cancer cells OVCAR-3. This indicates that the folic acid-conjugated, doxorubicin-loaded HCRSV capsid has potential for the targeted delivery of cancer chemotherapeutics. However, much work remains to be done to develop the protein cages of plant viruses into feasible drug delivery systems. For the fPC-Dox, further work on the immune response and in ViVo efficacy of the formulation will be required to assess its potential for clinical applications.

ACKNOWLEDGMENT This work was supported by the ARF grants R-148-000-045112 and R-154-000-252-112 from National University of Singapore (NUS). Ren Yupeng is a recipient of the NUS graduate research scholarship.

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Folic Acid-Conjugated Protein Cages of a Plant Virus - American

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