Improved drug targeting of cancer cells by utilizing actively targetable folic acid-conjugated albumin nanospheres

Pharmacological Research 63 (2011) 51–58

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Improved drug targeting of cancer cells by utilizing actively targetable folic acid-conjugated albumin nanospheres Zheyu Shen a,b,∗ , Yan Li c , Kazuhiro Kohama b , Brian Oneill a , Jingxiu Bi a,∗∗ a b c

School of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma 371-8511, Japan Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University, 11 Fucheng Road, Haidian District, Beijing 100037, China

a r t i c l e

i n f o

Keywords: Actively targetable Folic acid Albumin nanospheres Drug targeting Cancer cells

a b s t r a c t Folic acid-conjugated albumin nanospheres (FA-AN) have been developed to provide an actively targetable drug delivery system for improved drug targeting of cancer cells with reduced side effects. The nanospheres were prepared by conjugating folic acid onto the surface of albumin nanospheres using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as a catalyst. To test the efficacy of these nanospheres as a potential delivery platform, doxorubicin-loaded albumin nanospheres (DOX-AN) and doxorubicin-loaded FA-AN (FA-DOX-AN) were prepared by entrapping DOX (an anthracycline, antibiotic drug widely used in cancer chemotherapy that works by intercalating DNA) into AN and FA-AN nanoparticles. Cell uptake of the DOX was then measured. The results show that FA-AN was incorporated into HeLa cells (tumor cells) only after 2.0 h incubation, whereas HeLa cells failed to incorporate albumin nanospheres without conjugated folic acid after 4.0 h incubation. When HeLa cells were treated with the DOX-AN, FA-DOX-AN nanoparticles or free DOX, cell viability decreased with increasing culture time (i.e. cell death increases with time) over a 70 h period. Cell viability was always the lowest for free DOX followed by FA-DOX-AN4 and then DOX-AN. In a second set of experiments, HeLa cells washed to remove excess DOX after an initial incubation for 2 h were incubated for 70 h. The corresponding cell viability was slightly higher when the cells were treated with FA-DOX-AN or free DOX whilst cells treated with DOX-AN nanoparticles remained viable. The above experiments were repeated for non-cancerous, aortic smooth muscle cells (AoSMC). As expected, cell viability of the HeLa cells (with FA receptor alpha, FR␣) and AoSMC cells (without FR␣) decreased rapidly with time in the presence of free DOX, but treatment with FA-DOX-AN resulted in selective killing of the tumor cells. These results indicated that FA-AN may be used as a promising actively targetable drug delivery system to improve drug targeting to cancer cells. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Chemotherapy is a major therapeutic approach for the treatment of a wide range of cancers. A major problem with such treatment is the side effects as the agents affect healthy tissue as well as the cancerous cells. Hence, any selective increase in tumor tissue uptake would be a significant development in cancer therapy given the non-selectivity of the normally utilized drugs. This study is focused on an improved targeted treatment to deliver conventional anti-cancer drugs by utilizing the folic acid receptor alpha (FR␣). This receptor alpha, a glycosylphosphatidylinositol-linked protein, is over-expressed on the surface of numerous human

∗ Corresponding author at: School of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia. Tel.: +61 883036912; fax: +61 883034373. ∗∗ Corresponding author. Tel.: +61 883034118; fax: +61 883034373. E-mail addresses: [email protected] (Z. Shen), [email protected] (J. Bi). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.10.012

cancer cells (including the malignant tumor cells of ovary, brain, kidney, breast, lung and uterine cervix) [1]. Whereas, this protein is seldom present on the surface of normal healthy cells [2], this difference can potentially be exploited to target the malignant cells for improved drug delivery. Unfortunately, the precise mechanism of FR␣ transport of folic acid (FA) into cancer cells is unresolved but it is clear that these folic acid conjugates may be nondestructively taken up by cancer cells via receptor-mediated endocytosis [3,4]. As a consequence of this observation, FA has been applied to provide a targeting moiety for anticancer drug carriers to minimize non-specific attack on normal cells and to increase cellular uptake by the target tumor cells thereby improving the therapeutic efficacy of anticancer drugs [5,6]. Several FA-conjugated polymeric micelles have been observed to display higher cytotoxicities and cellular uptake for FR␣-positive cancer cells [7–10]. This application of FA is very attractive given its low immunogenicity, low cost, ease of modification, small size (Mw = 441.4), stability during storage, compatibility

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Scheme 1. A schematic summarizing the preparation strategy for the production of FA-DOX-AN. The small spheres represent the DOX; the large spheres represent the AN; lines in the large spheres represent crosslinkage by glutaraldehyde.

with a variety of organic and aqueous solvents, and high binding affinity (Kd ≈ 10−10 M) with the FR␣ receptor [11]. However, two disadvantages are associated with the use of FA-conjugated polymeric micelles: first, FA-conjugated polymeric micelles are inherently unstable as stability is primarily provided by rather weak hydrophobic interaction, whereas covalent bonding would provide stronger bonding forces [12]; second, FA-conjugated polymeric micelles may only be used as a reservoir and delivery system for water-insoluble anticancer drugs given their hydrophobic inner core. This is a serious limitation as numerous anticancer drugs are water-soluble [13,14]. Doxorubicin (an anthracycline antibiotic) is an anti-cancer (“antineoplastic” or “cytotoxic”) chemotherapy drug. Its mode of action is complex and not completely understood but it is believed to interact with cell DNA by intercalation and subsequent inhibition of biosynthesis. It induces a wide variety of undesirable side effects including nausea, vomiting, pain at the injection site, hair loss, reduction in white blood cells and heart arrhythmia. It was used as the chemotherapeutic agent in this work. The aim of this study was to overcome these identified disadvantages of FA-conjugated polymeric micelles and to develop an actively targetable drug delivery system using folic acid-conjugated albumin nanospheres (FA-AN). At a first step, the biodegradable albumin nanospheres were prepared by using a desolvation technique coupled with chemical cross-linking by glutaraldehyde. Next, the folic acid was conjugated onto the surface of the nanospheres in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). Finally, doxorubicin-loaded AN (DOX-AN) and doxorubicin-loaded FA-AN (FA-DOX-AN) were prepared by entrapping doxorubicin (DOX) into the nanospheres during the particle preparation. A schematic summarizing the process steps in the preparation sequence is presented as Scheme 1. The conjugated nanoparticles prepared in this manner possess following attributes: first, their three dimensional structure is stabilized by strong covalent bonding (cross-linking by glutaraldehyde) rather than relying on weaker hydrophobic interaction; second, FA-AN may be utilized as a delivery vehicle for watersoluble drugs due to the presence of hydrophilic albumin; finally,

cross-linking of the nanospheres with glutaraldehyde provides stability and enhanced potential for biological tracing and quantitative analysis studies as a consequence of increased auto-fluorescence. Given these attributes, folic acid conjugated albumin nanoparticles should provide us a promising actively targetable drug delivery system. 2. Materials and methods 2.1. Materials Bovine serum albumin (BSA, fraction V, pH 7.0), l-lysine (Lys), EDAC, FA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and hoechst33258 were purchased from Sigma (USA). A 50% glutaraldehyde aqueous solution was supplied by Kanto Chemical Co., Inc. From this solution, an 8% glutaraldehyde aqueous solution was prepared by dilution with MilliQ water. Trypsin (≥250 NF U/mg) was ordered from Nippon Becton Dickinson Co., Ltd. (Tokyo, Japan). Doxorubicin was supplied by TRC (Toronto Research Chemical). Rhodamine-phalloidin (RP) was purchased from Molecular Probes (Eugene, OR). All reagents were of analytical grade and used without further purification. 2.2. Preparation of blank and DOX-loaded albumin nanospheres Albumin nanospheres were prepared using a modified desolvation technique coupled with chemical cross-linking by glutaraldehyde [12]. Briefly, 2.0 mL of ethanol (the desolvation agent) was added dropwise into a 2.0 mL aqueous solution of BSA (20 mg/mL dissolved in a 10 mM NaCl solution at pH = 10.8) under magnetic stirring. Next, 4.0 mL of ethanol was continuously added at a rate of 2.3 mL/min at room temperature again with magnetic agitation at 600 rpm. Following this ethanol addition step, 60 ␮L of an 8% aqueous solution of glutaraldehyde was rapidly added to induce particle cross-linking. Completion of the cross-linking process required stirring of the suspension for 24 h. Then, 1.0 mL of an aqueous solution of Lys (40 mg/mL) was added to cap the free aldehyde groups. After allowing this reaction to proceed for 2.0 h,

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Table 1 Preparative conditions and results of AN and FA-AN. Sample

Conjugating time (h)

Yield (%)a

Mean particle size (nm)

PDIb

AN FA-AN1 FA-AN2 FA-AN3 FA-AN4 FA-AN5 DOX-AN FA-DOX-AN1 FA-DOX-AN4

– 4.0 8.0 12 16 24 – 4.0 16

73 76 84 81 72 89 77 78 83

151.1 160.9 158.5 163.1 160.6 188.8 199.3 176.2 191.6

0.097 0.175 0.107 0.092 0.079 0.205 0.193 0.097 0.168

a b c

Zeta potential (mV) −20.2 −23.8 −23.9 – −24.0 −26.5 – – –

Mass conjugated (nmol/mg NS)c

DLC (%)c

DLE (%)c

– 6.42 18.3 23.0 25.9 26.9 – 3.94 20.9

– – – – – – 2.00 1.85 1.76

– – – – – – 60.1 55.8 52.8

Calculated from the weight ratio of nanospheres to feed materials. Polydispersity index. Determined by UV spectrophotometer and calculated from the corresponding standard calibration curve; DLC: drug loading content; DLE: drug loading efficiency.

the suspension was centrifuged (at 30,000 × g, for 20 min) at 15 ◦ C (Himac CP100WX Preparative Ultracentrifuge, HITACHI) to produce the albumin nanospheres. The harvested nanospheres were washed twice with MilliQ water to remove any remaining traces of the non-desolvated BSA as well as any excess of Lys. Following each washing stage, the nanospheres were centrifuged and supernatant was discarded. Finally, the nanoparticles were lyophilized for 48 h (Eyela Freeze Dryer FD-1). DOX-AN nanospheres were prepared using an identical method with 500 ␮g/mL DOX dissolved in 20 mg/mL BSA aqueous solution. A summary of the preparative conditions and the results is provided in Table 1. 2.3. Conjugation of FA onto the surface of AN and DOX-AN FA-AN and FA-DOX-AN nanoparticles were prepared by conjugating folic acid onto the surface of AN and DOX-AN, respectively. Typically, 30.0 mg of EDAC was dissolved in 1.0 mL of ice cold FA solution (500 ␮g/mL in PBS). Then 9.0 mL of AN (or DOX-AN) suspensions (1.0 mg/mL in PBS) was added and the mixture was stirred at room temperature for residence times varying from 4.0 to 24 h. Next, the resulting FA-AN or FA-DOX-AN nanoparticles were ultra-centrifuged and the supernatants were stored for further analysis. Harvested samples were then washed with MilliQ water and lyophilized for 48 h. The unreacted folic acid remaining in the supernatant was quantified using a UV-spectrophotometer (HITACHI, U-2000). The absorbance (wavelength = 363 nm) of the supernatant was converted into a folic acid concentration by using a calibration curve constructed with standard FA solutions. A simple mass balance was then used to calculate the FA conjugation on the surface of the nanospheres. Preparative conditions and the results are summarized in Table 1. 2.4. TEM observation and dynamic light scattering measurement of nanospheres The resulted particles of AN, FA-AN, DOX-AN and FA-DOXAN were observed using a JME-1010 (JEOL, Japan) transmission electron microscope (TEM). Approximately 3.0 ␮L of the diluted solution of the nanospheres was mounted on Formvar coated copper grids and then dried at room temperature. A 10% alcoholic solution of uranyl acetate was prepared and centrifuged to remove any precipitate. The sections on copper grids were stained for 15 s with uranyl acetate in the dark at room temperature. Finally, the samples were dried at room temperature, then observed using the electron microscope (transmission mode). The size distributions and zeta potentials of the nanospheres in phosphate buffered solution (PBS) at room temperatures were measured by Zetasizer Nano Particle Size Analyzer (Nano-ZS ZEN3600, Malvern, UK). The solutions were optically cleaned by passage through a 0.45 ␮m membrane filter prior to measurement.

Dynamic light scattering (DLS) histograms were calculated from the autocorrelation function of the scattered light intensity using propriety Dispersion Technology Software (Nano series and HPPS, DTS v4.20) based on a Non-Negative Least-Squares (NNLS) algorithm [15]. The particle size and polydispersity index (PDI) were calculated by the cumulant method. In this method, the electric field correlation function g(1) () is expressed in a form of an expansion as,



g (1) () = exp −1  +

2 2  − ··· 2!



(1)

where  i is the ith cumulant. The z-average diffusion coefficient D was calculated from the first cumulant as  1 = Dq2 , where q is the scattering vector, and the average particle size was estimated from the diffusion coefficient D by using the well-known Stokes–Einstein equation. PDI of the particles was obtained as 2 /12 . In the stability study, AN were suspended in the saline buffer (PBS, 0.2 mg/mL) at 4 ◦ C and the particle size following storage was monitored using dynamic light scattering. 2.5. Determination of drug loading content and drug loading efficiency The doxorubicin content of the particles was measured by digesting 10 mg of DOX-AN (or FA-DOX-AN) and a corresponding blank AN (or blank FA-AN) in 6 mL of trypsin solution (10 mg/mL) in the dark for 4.0 h at 37 ◦ C, respectively. The digested solution of blank AN (or blank FA-AN) was used to produce a baseline of absorbance measurement by UV spectrophotometer (HITACHI, U2000). The absorbance (at 480 nm of wavelength) of the digested DOX-AN (or FA-DOX-AN) solution was converted into the DOX concentration using the calibration curve constructed with standard DOX solutions containing 1.67 mg/mL of BSA and 10 mg/mL of trypsin. The drug loading content (DLC, %) and drug loading efficiency (DLE, %) were calculated from the following formulae: DLC (%) =

weight of DOX in AN × 100 weight of AN

(2)

DLE (%) =

weight of DOX in AN × 100 weight of the feeding DOX

(3)

2.6. In vitro release profiles of DOX from the prepared nanospheres The release of doxorubicin from DOX-AN (or FA-DOX-AN) with or without 2.0 mg/mL of trypsin was determined at 37 ± 1 ◦ C. Typically, 30 mg of DOX-AN (or FA-DOX-AN) was dispersed in 20 mL of PBS with or without 2.0 mg/mL of trypsin. The suspensions were incubated (Incubator IC63, Yamato, Tokyo, Japan) at 37 ± 1 ◦ C with shaking at 120 rpm (Recipro Shaker NR-10, Taitec, Saitama). At pre-

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determined time intervals, these samples were ultracentrifuged at 30,000 × g for 20 min. A 1.0 mL sample from these supernatants was collected (1.0 mL of PBS was supplemented) and analyzed to determine the released DOX concentration using a spectrophotometer (wavelength = 480 nm). 2.7. Cell cultures The human cervix carcinoma cell line, HeLa (kindly supplied by Dr Wang, Gunma University Graduate School of Medicine, Gunma, Japan) and the human aortic smooth muscle cell line, AoSMC (purchased from Lonza, Walkersville, MD, USA) were incubated (37 ◦ C, 5% CO2 ) in a high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, including 100 ␮g/mL of streptomycin and 100 IU/mL penicillin. 2.8. Cell uptake of the prepared nanospheres Uptake of the AN or FA-AN nanospheres by the HeLa cells was investigated using a laser scanning confocal microscopy (LSCM) (Zeiss M510 Meta). 2.0 mL of HeLa cells were seeded onto a cell culture dish (Ø40 mm × 10 mm) with a cell density of 1.0 × 104 cells/mL and allowed to adhere overnight. The growth medium was replaced with a fresh medium (2.0 mL) containing 0.502 mg/mL (determined by fluorescence spectrophotometry following centrifugation of the nanoparticles at 2000 × g for 5.0 min to remove aggregates) of AN (or FA-AN4). Then cells were incubated at 37 ◦ C for 4.0 h (or 2.0 h). At the conclusion of this period, the cell layer was washed thrice with PBS solution. The cells were then fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 5.0 min, and blocked with 1.0% BSA for 30 min at room temperature prior to imaging in the LSCM. Actin present in cells was then stained with 3–5 U/mL Rhodamine-phalloidin (RP) for 30 min. The cells were washed thrice with PBS between each step. 2.9. Quantitative analysis of the internalized nanospheres by cells The quantity of nanospheres incorporated into the HeLa cells was determined using a fluorescence spectrophotometer (F-4500, HITACHI). Typically, 7.0 mL of HeLa cells were seeded onto a cell culture dish (Ø90 mm × 20 mm) with a density of 5.0 × 105 cells/mL and allowed to adhere overnight. Next, the growth medium was replaced with fresh medium (7.0 mL) containing 0.05–0.50 mg/mL AN (or FA-AN4) and the cells were then incubated for periods ranging from 1.0 to 12 h. Upon completion of this growth phase, the cells were washed twice with PBS, treated with trypsin for 3.0 min and then centrifuged at 2000 × g for 5.0 min. The resulting cells pellet was resuspended in 1.0 mL of DMSO. Finally, the samples were excited at 490 nm and the fluorescence intensity at a wavelength of 516 nm was determined using a fluorescence spectrophotometer. The fluorescence intensity of the samples was converted to a mass of nanospheres using a calibration curve constructed with standard AN (or FA-AN4) solutions. 2.10. Viability of cells treated with distinct DOX formulations The cytotoxicity of the various DOX formulations was measured using the MTT Proliferation Assay. Yellow MTT is reduced to purple formazan in living cell’s mitochondria. The absorbance of this solution can be quantified by spectrophotometry. This reduction only occurs if mitochondrial reductase enzymes are active, thus conversion is directly related to the number of viable cells. Typically, 150 ␮L of HeLa (or AoSMC) cells were seeded in 96-well plates at a density of 1.0 × 105 cells/mL and allowed to adhere overnight. The

growth medium was replaced with fresh medium (200 ␮L) containing 5.0 ␮g/ml of the DOX formulations investigated in this study: namely, AN, FA-AN4, DOX-AN, FA-DOX-AN4 and DOX. Cells were then incubated for periods ranging from 3.0 to 70 h; or alternatively, cells were washed with PBS after a brief incubation of 2.0 h, then 200 ␮L of fresh growth medium was added and the cells were subsequently incubated for 1.0–68 h. Next, 10 ␮L of MTT (5.0 mg/mL in PBS) was added and cells were incubated for an additional 4.0 h at 37 ◦ C. After that, the growth medium was removed and 150 ␮L of DMSO was added to each well to ensure solubilization of formazan crystals. Finally, the absorbance was determined using a multiwell scanning spectrophotometer (VMax Kinetic Microplate Reader with Softmax Pro from Molecular Devices) at a wavelength of 570 nm. To eliminate any effects from drugs incorporated into the cells on absorbance measurement, an identical experiment without MTT (with all other conditions fixed) was carried out in 96-well plates and the corresponding wells (after addition of DMSO) were used to prepare the baseline for subsequent absorbance measurement, respectively. 2.11. Statistical analysis Statistical significance was determined by application of Student’s t-test or by a one-way ANOVA followed by Student–Newman–Keuls test using Sigma Stat version 3.5. The differences were considered significant for P < 0.05. 3. Results and discussion 3.1. Characteristics of the prepared nanospheres by TEM and DLS The nanospheres prepared using the desolvation technique and modified with folic acid were initially analyzed using TEM. Fig. 1(a)–(d) presents a series of TEM photographs (observed at 120,000 times magnification) of the AN, FA-AN4, DOX-AN and FADOX-AN4 nanoparticles, respectively. It is clear that the resulting nanospheres are spherical in shape and uniform in size. As well, the nanospheres are individually dispersed and the level of coalescence and aggregation is minimal. Unfortunately, folic acid attached to the surface of albumin nanospheres is not visible when using TEM. This result was expected given the low molecular weight of the folic acid molecule (441.40 Da). Particle size, size distribution and the zeta potential are likely to play important roles in determining the nanoparticles’ fate following intravenous administration [16]. Previous studies have reported that phagocytosis of the tumor cells is dependent on the sizes of nanospheres [17]. Typically, nanospheres with diameters lying between 100 and 300 nm accumulate preferentially in tumor tissue rather than in normal tissues. This effect appears to be a consequence of increased permeability of the tumor vasculature and ineffective lymphatic drainage (EPR effect) [17,18]. In this study, the particle size, size distribution and the zeta potential of the nanospheres (with or without folic acid conjugation) were determined by dynamic light scattering (DLS). As shown in Table 1, the mean particle size of the prepared nanospheres was below 200 nm. All observed size distributions Fig. 1(e)–(h) were narrow (previously confirmed by TEM). Thus, it is reasonable to suggest that these albumin nanospheres should provide a potentially attractive drug delivery scheme. Active cellular targeting may be achieved by functionalizing the surface of nanospheres with ligands that promote cell-specific recognition and binding. The nanospheres should be capable of (i) releasing their contents in close proximity to the target cells; (ii) attaching to the cell membrane then acting as an extracellular sustained-release drug

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Fig. 1. TEM photo of AN (a), FA-AN4 (b), DOX-AN (c), and FA-DOX-AN4 (d) observed at 120,000× magnification. Corresponding size distribution of nanospheres for AN (e), FA-AN4 (f), DOX-AN (g), and FA-DOX-AN4 (h).

depot; or (iii) being incorporating into the cell [18]. Hence, FAAN should provide a promising actively targetable drug delivery system. As well, the prepared nanospheres possess a negative surface charge due to the carboxyl groups on the hydrophilic shell of nanospheres. The measured zeta potential of prepared nanospheres (Table 1) were −20.2 mV, −23.8 mV, −23.9 mV, −24.0 mV and −26.5 mV for AN, FA-AN1, FA-AN2, FA-AN4 and FA-AN5, respectively. Cellular uptake of FA-AN is unlikely to be reduced by the electrostatic repulsion force which exists between the nanospheres and the negative surface charge of cells. That’s because specific receptor-mediated interactions may overcome the repulsive effect and control the rate of nanospheres uptake into the target cells. Furthermore, the anionic carriers are preferred for the selective delivery of drugs to the cells, as cationic carriers may lead to non-specific binding to a various of cells after systemic administration [17]. Additionally, the zeta potential of nanospheres decreased following folic acid conjugation due to conjugation of folic acid to the amino groups (–NH2 ) of the nanoparticles thereby reducing the positive surface charge. During storage, aggregation of nanospheres may occur resulting in a loss of structural integrity or the formation of inactive

precipitates [19]. Clearly, the stability of nanospheres during storage must be evaluated, consequently a detailed study of stability over time was performed. Neither aggregation nor precipitation of the nanospheres was observed during storage periods of up to one month. This beneficial effect is a likely consequence of electrostatic repulsion between the negatively charged nanospheres. As well, no change in particle size of the nanospheres was observed throughout the entire course of the study (Fig. 2). These results confirm that preparations of the nanospheres (suspended in PBS) should exhibit a long shelf life.

3.2. Conjugating result of folic acid onto albumin nanospheres As noted previously, folic acid (with carboxyl groups) was conjugated onto the surface of AN and DOX-AN (with amino groups) nanoparticles in the presence of EDAC. Table 1 presents a summary of preparative conditions and results. The mass of FA on the surface of nanospheres (NS) was determined to be 6.42, 18.3, 23.0, 25.9, 26.9 nmol/mg NS for FA-AN1 to FA-AN5 and 3.94, 20.9 nmol/mg NS for FA-DOX-AN1, FA-DOX-AN4, respectively.

Mean Diameter (nm)

200

180

160

140

120 5

10

15

20

25

30

Storage Time (days) Fig. 2. Change in mean particle size of the AN (0.2 mg/mL in PBS) during storage at 4 ◦ C (mean ± SD, n = 3).

Fig. 3. Influence of the conjugating time on the amount of FA attached to the surface of the albumin nanospheres.

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DOX cumulative release (%)

80

60

DOX-AN FA-DOX-AN4

40

DOX-AN, Trypsin FA-DOX-AN4, Trypsin 20

0 60

90

120

150

Time (hours) Fig. 4. In vitro release profiles of DOX from the DOX-AN or FA-DOX-AN4 in PBS with or without 2.0 mg/mL trypsin at 37 ◦ C.

The number of FA molecules attached on the surface of a single nanosphere may be calculated as: 4  3

 3 d¯ 2

 =n×



4 d0  3 2

3 (4)

where d¯ is the mean diameter of nanosphere (NS),  is the volume ratio of BSA molecules in one NS, n is the number of BSA molecules in one NS, and d0 is the mean diameter of BSA. d0 was determined to be 5.1 nm using DLS at room temperature with a concentration of 5.0 mg/mL. Assuming that  ≈ 1.0 (as the particle is primarily composed of BSA), it can be determined that nAN = 26, 000 due to d¯ AN = 151.1 nm. The corresponding value of nDOX-AN = 59, 700 as d¯ DOX-AN = 199.3. In molar units this represents 5.83 × 10−13 mol within 1.0 mg AN and 2.54 × 10−13 mol within 1.0 mg DOX-AN (assuming 1.0 mg NS is consisted of 1.0 mg BSA). Therefore, the number of folic acid on the surface of NS may be calculated to be 1.1, 3.1, 3.9, 4.4, 4.6 × 104 mol/mol NS for FAAN1 to FA-AN5 and 1.6, 8.2 × 104 mol/mol NS for FA-DOX-AN1, FA-DOX-AN4, respectively. The conjugating amount of FA on the surface of AN was obviously influenced by the conjugating time as shown in Fig. 3. The conjugating amount of FA initially increased rapidly with increasing the conjugating time. However, it was significantly close when the conjugating time was longer than 16 h. As shown in Table 1, the drug loading content (DLC, %) and drug loading efficiency (DLE, %) of both FA-DOX-AN1 and FA-DOXAN4 were lower than those for DOX-AN. This difference may be attributed to a loss of drug during the conjugation process. Moreover, possible explanations for the high DLEs (greater than 50%, Table 1) are: (1) DOX is readily entrapped in albumin nanospheres due to (i) hydrophobic interaction between DOX and albumin, and (ii) ionic interaction between the –NH3 + of DOX and –COO− of albumin; and (2) DOX is not readily released from albumin nanospheres during washing process due to chemical crosslinkage of the nanospheres by glutaraldehyde.

Fig. 5. Laser scanning confocal microscope images of HeLa cells incubated with AN or FA-AN4 at the indicated time. The LSCM images of the first column are for HeLa cells incubated with AN; the second column is HeLa cells incubated with FA-AN4.

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3.3. In vitro release profiles of DOX from DOX-AN or FA-DOX-AN Fig. 4 summarizes the release profiles of DOX from the prepared nanospheres with and without 2.0 mg/mL trypsin in PBS at 37 ◦ C. In the absence of trypsin, both DOX-AN and FA-DOXAN exhibit a similar release profile of DOX. Roughly 5% of the loaded drug in the DOX-AN or FA-DOX-AN particles is released after 5 h. A likely explanation of this high value is that drug material attached at or near the outer shell of nanospheres is not tightly bound and consequently will be easily released. As shown after 160 h, a total 13% of the loaded drug was released, this significantly reduced delivery rate suggests that the release of DOX from DOX-AN and FA-DOX-AN may be sufficiently slow so as to minimize harmful side effects of DOX. In the presence of trypsin, the release of DOX from both DOX-AN and FA-DOX-AN accelerated dramatically. If the nanoparticles are to serve as a practical anti-cancer drug carrier for tumor targeting, then a slow drug release rate is desirable as the damage from side effects of potent anti-cancer drugs will be minimized during circulation in the patient’s blood stream. As well, the release rate should be accelerated at the target sites (cancer cells or cancer tissues) where enzymes are more abundant than blood cells [12]. Clearly, the observed, reduced release rate of DOX from the prepared nanospheres in the absence of trypsin and the dramatically increased release in the presence of trypsin suggest that both AN and FA-AN should be effective potential anti-cancer drug carriers with reduced damaging side effects. 3.4. Cellular uptake of the prepared nanospheres HeLa cells are known to recognize FA via the FR␣ receptor attached to their surface. Once a ligand binds to the FR␣ receptor, the ligand–receptor complex is rapidly incorporated into the HeLa cells and the receptor then recycles back to the cells’ surface [8]. To assess the extent of incorporation of the prepared nanospheres, HeLa cells were incubated with either AN or FA-AN4 nanoparticles. The AN and FA-AN4 cross-linked with glutaraldehyde (showed autofluorescent property) were yellow whilst actin present in the cells stained with RP was red (Fig. 5). It is clear from the photo that FAAN4 enters into the HeLa cells after 2.0 h incubation, whereas AN is not incorporated into the HeLa cells even after 4.0 h incubation. After qualitative confirmation of this difference by LSCM, the level of AN and FA-AN4 present in the HeLa cells was determined by fluorescence spectrophotometer. The mass of FA-AN4 in the HeLa cells is significantly greater than that for AN (Fig. S1, P < 0.01). As well, the quantity of incorporated FA-AN4 by cells increased with increasing the incubation time in 4.0 h, and increased with increasing the concentration of FA-AN4 in 0.5 mg/mL (Fig. S2). These results indicated that FA-AN prepared in this study have a specific affinity for the cancerous HeLa cells via ligand–receptor (FA-FR␣) recognition. The presence of AN in the HeLa cells is clearly shown in Fig. S1, however it was not observed by LSCM as evidenced in Fig. 5. That’s because nanospheres adhering to the surface cells are difficult to remove by washing with PBS and consequently some particles remain attached to cells and are precipitated during centrifugation. 3.5. Viability of HeLa cells treated with distinct DOX formulations Finally, the killing efficacy of various DOX formulations on HeLa (or AoSMC) cells was investigated in this study. Fig. 6 presents the results showing the effect of culture time on the viability of HeLa cells treated with AN, FA-AN4, DOX-AN, FA-DOX-AN4 or DOX (CDOX = 5.0 ␮g/mL). Three scenarios were studied as follows: Fig. 6(a) the various DOX formulations were continuously

Fig. 6. Cytotoxicity of HeLa cells (with FR␣) or AoSMC cells (without FR␣) treated with distinct DOX formulations (CDOX = 5.0 ␮g/mL). (a) DOX formulations were continuously incubated with HeLa cells; #P < 0.01 compared with FA-DOX-AN4. (b) DOX formulations were washed out by PBS after 2.0 h of incubation with HeLa cells; #P < 0.001 compared with FA-DOX-AN4. (c) DOX formulations were washed out by PBS after 2.0 h of incubation with AoSMC cells (mean ± SD, n = 4).

incubated with HeLa cells for periods from 3.0 to 70 h; Fig. 6(b) summarizes the results for the same range of total incubation periods with the DOX formulations washed from the cells using PBS after 2.0 h of incubation; and Fig. 6(c) repeats the procedure in (b) with AoSMC cells. As shown in Fig. 6(a), cell viability remains constant at roughly 100% (as expected) when the HeLa cells were treated with nanoparticles containing no toxic agent (i.e. AN or FA-AN4). Introduction of the DOX causes a reduction in cell viability with increasing the culture time (i.e. DOX-AN, FA-DOX-AN4 or DOX). The order of efficacy as a killing agent is DOX, then FA-DOX-AN4 and finally DOX-AN. Fig. 6(b) presents the time course of cell viability with washing out the DOX formulations after 2.0 h of incubation with HeLa cells. Compared to results in Fig. 6(a), the corresponding cell viability is comparable when cells were treated with AN or FAAN4 (as expected), it is slightly higher when cells were treated with FA-DOX-AN4 or free DOX, whereas it significantly increased when cells were treated with DOX-AN. Fig. 6(c) presents result for AoSMC cells using an identical protocol to that in Fig. 6(b). Compared with Fig. 6(b), the corresponding cell viability is very similar when the cells were treated with DOX-AN or free DOX, whereas it is significantly increased when the cells were treated with FA-DOX-AN4. Clearly, the nanospheres alone (AN and FA-AN) exhibit no cytotoxicity towards HeLa cells, suggesting excellent biocompatibility. By contrast, FA-DOX-AN is significantly slower in inhibiting growth

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of HeLa cells when compared with free DOX. This result is expected due to rapid diffusion of free DOX into HeLa cells versus a sustained release of DOX from FA-DOX-AN (P < 0.001, n = 4). FA-DOX-AN was more effective in inhibiting growth of HeLa cells than DOX-AN as a result of rapid incorporation into HeLa cells via the specific ligand–receptor (FA-FR␣) interaction (P < 0.01, n = 4). Washing DOX formulations after 2.0 h of incubation was undertaken for the HeLa & AoSMC cells. The cell viability was only slightly higher than the result of the continuously incubated case (i.e. HeLa cells were treated with FA-DOX-AN or free DOX). This suggests that significant amounts of FA-DOX-AN or free DOX were incorporated into HeLa cells only after 2.0 h of incubation. Cell viability was significantly improved for treatment with DOX-AN suggesting that it is not readily incorporated after 2.0 h of incubation. Finally, the washing procedure following a short incubation time (Treatment 6(b)) was repeated with AoSMC cells rather than tumor cells and the results are summarized in Fig. 6(c). Comparing with HeLa cells, the corresponding cell viability was very close when cells were treated with DOX-AN or free DOX because DOX-AN and free DOX were nonselective to both HeLa cells (with FR␣) and AoSMC cells (without FR␣), and it was very higher when the cells were treated with FA-DOX-AN because FA-DOX-AN was selective to HeLa cells and nonselective to AoSMC cells. 4. Conclusions In this work, folic acid conjugated albumin nanospheres have been developed by attaching folic acid onto the surface of the nanospheres. The resultant particles are spherical in shape, uniform in size with a mean diameter below 200 nm. The molar ratio of FA conjugated onto the surface of nanoparticles was 4.6 × 104 mol/mol albumin nanoparticles. The resultant particles (FA-DOX-AN) possess attractive characteristics that suggest their use as a vehicle for efficient drug delivery and targeting. First, the slow release rate of the well known chemotherapic agent DOX from the prepared nanospheres in the absence of the trypsin contrasted with obvious high release in the presence of trypsin suggest that they may be used to mitigate the harmful side effect of anti-cancer drugs. As well, the FA-AN nanoparticles are readily incorporated into HeLa cells after a short incubation period (2.0 h), whereas AN alone is almost not incorporated into HeLa cells even if the incubation period doubles to 4.0 h. As expected, the FA-DOX-AN nanoparticles displayed more potency in inhibiting the growth of HeLa cells when compared to that for the DOX-AN particles, especially if the DOX formulations were removed by washing out with PBS after 2.0 h incubation with cells. AoSMC cell viability was higher when compared to viability of HeLa cells if the cells were treated with FA-DOX-AN, suggesting that FA-DOX-AN was selective for HeLa cells (possessing the surface FR␣ receptor) and non-selective for AoSMC cells (without the FR␣ receptor). These results confirm the hypothesis that FA-AN nanoparticles should provide an actively targetable drug delivery system to improve drug targeting to cancerous cells.

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