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Antitumor activity of galactoxyloglucan-gold nanoparticles against murine ascites and solid carcinoma
Colloids and Surfaces B: Biointerfaces 116 (2014) 219–227
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Antitumor activity of galactoxyloglucan-gold nanoparticles against murine ascites and solid carcinoma Manu M. Joseph a , S.R. Aravind a , Suraj K. George b , K. Raveendran Pillai c , S. Mini d , T.T. Sreelekha a,∗ a
Laboratory of Biopharmaceuticals and Nanomedicine, Division of Cancer Research, Regional Cancer Centre, Trivandrum, Kerala, India Dept. of Hematopathology, UT MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, 77030, TX, USA c Department of Pathology, Regional Cancer Centre, Trivandrum, Kerala, India d Department of Biochemistry, University of Kerala, Trivandrum, Kerala, India b
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 2 December 2013 Accepted 27 December 2013 Available online 9 January 2014 Keywords: Anticancer Dalton’s Lymphoma Ascites Ehrlich’s Ascites Carcinoma Polysaccharide Galactoxyloglucan Gold nanoparticle Tamarindus indica
a b s t r a c t Galactoxyloglucan polysaccharide (PST001), isolated from the seed kernels of Tamarindus indica (Ti), was used both as reducing and capping agent for the preparation of gold nanoparticles (PST-Gold) of 20 nm size. The present study evaluated the anticancer effects of the PST-Gold nanoparticles both in vitro and in vivo. The cytotoxicity was evaluated in the murine cancer cell lines, Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC). Galactoxyloglucan-gold nanoparticles (PST-Gold) not only retained the anticancer effects of PST001, but also showed enhanced cytotoxicity via induction of apoptosis even at lower doses and lesser incubation times. In vivo antitumor activity was tested in DLA and EAC murine ascites and EAC solid-tumor syngeneic mouse models. PST-Gold nanoparticles reduced tumor burden and increased median survival and life span significantly in both tumor models compared to the controls. The PST-Gold nanoparticles were very effective as a chemopreventive agent, showing the best overall response when administered prior to tumor induction. In the case of solid tumors, intratumoral administration of the PST-Gold nanoparticles yielded significant results with regard to survival and increment in lifespan as compared to intraperitoneal mode of drug administration. Further studies in higher animal models and in patients at high-risk for recurrence are warranted to fully explore and develop the potential of PST-Gold nanoconjugates as a chemopreventive and therapeutic anti-cancer agent. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a major cause of mortality worldwide and, in spite of the extraordinary effort and money spent over the past several decades, successful eradication and control remains intangible [1]. Cancer is described as a heterogeneous collection of cells that evolve in tumor microenvironments with complex ecologies [2]. The rapid progression of human cancers is related to the evasion of the body’s immune surveillance mechanisms by tumor cells through the secretion of immunosuppressive factors that modify the host immune response. Currently available chemotherapeutic modalities also suppress the immune system, predisposing cancer patients to secondary infections [3]. In this regard, there is a
∗ Corresponding author at: Scientific Officer Grade 1, Division of Cancer Research, Regional Cancer Centre (RCC), Trivandrum-11, Kerala, India. Tel.: +91 4712522378; fax: +91 4712447454. E-mail addresses: [email protected], [email protected], [email protected] (T.T. Sreelekha). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.058
significant unmet need for novel pharmaceutical agents with tumor selectivity and specificity, but with limited side effects. Tamarindus indica (Ti), a tree of the Leguminosae family, is widely grown in India and is associated with numerous beneficial effects. Previous studies have shown the predominant composition of the Ti seed kernel to be a polysaccharide, galactoxyloglucan; ˛ (1 → 4)-- D-glucan backbone substituted with side chains of d-xylopyranose and -d-galactopyranosyl (1 → 2)-␣-dxylopyranose linked (1 → 6) to glucose residues [4,5]. Antioxidant, antitumor, immunomodulatory, antimicrobial, antiulcer, and several other pharmacological activities from various polysaccharides have been reported [6–8]. Among the biopharmacological properties of polysaccharides, immunomodulatory and antitumor effects are of high priority and the majority of these agents act as biological response modifiers, which enhance immunity [9]. Ti polysaccharides have widespread utility in anticancer, immunomodulatory, antiviral, antioxidant and other pharmacological applications including its use as artificial tears in modern medicine [10–12]. They are biocompatible, biodegradable, highly viscous, and thermally stable polymers with a broad range of pH tolerance and adhesiveness [13]. Galactoxyloglucan PST001, extracted from the
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seed kernels of Ti, was previously isolated and characterized in our laboratory [10]. Earlier studies have shown that PST001 leaves normal cells unharmed, while demonstrating significant anticancer and immunomodulatory properties [10,11,14]. However, due to the advent of gold nanoparticles, a new era of combination therapeutics has begun. Gold nanoparticles (AuNPs) have the advantage of delivering drugs specifically onto the target sites [15] with unique features, including tunable surface characteristics and microenvironment stability, along with negligible side effects. AuNPs accumulate in tumor specific sites which could be utilized to trace the path of cancer cells [16]. Even though the conventional methods for the synthesis of AuNPs involve secreting toxic chemicals to the environment, the ‘green chemistry’ approach using natural products have been reported to be devoid of significant biological hazards [17,18]. Among many other natural products with potential, polysaccharides represent excellent candidates for AuNP synthesis due to the presence of hydroxyl groups, a hemiacetal reducing end and other functionalities which could play important roles in the reduction and stabilization of AuNPs. Various polysaccharides such as chitosan, cellulose, sucrose and starch have been used for the synthesis and stabilization of AuNPs [19,20]. Preliminary studies have shown that the PST-Gold nanoconjugates exert significant in vitro cytotoxicity and in vivo immunostimulatory effects with scant toxic effects in normal tissues compared to their parental counterparts [21]. However, in vivo anticancer activities in more challenging ascites solid tumor models were not investigated, which is critical for the advancement of drug to clinical trials. Thus, herein, we examined the potential of PST-Gold nanoconjugates by which the immune system of the host could be modulated to enhance the anticancer effects in vitro and in vivo. Our data show that the nanoconjugates possess superior in vitro and in vivo cytotoxicity and immunomodulatory activities in both DLA and EAC tumors. 2. Materials and methods 2.1. Preparation and characterization of PST-Gold nanoparticles Ti seeds were obtained from reliable sources and was used to isolate and characterize PST001 in the first phase following the protocol previously reported [10]. The carbohydrate content was estimated by the standard Dubois’s method [22] utilizing d-glucose as the standard. In the second phase, PST-Gold nanoparticles were prepared and characterized as described previously [21]. In brief, 3 ml of 10 mg/ml solution of PST001 was added drop-wise to 1 ml solution of HAuCl4 (1 mM) with constant stirring on a magnetic stirrer; this was then placed in a hot plate heated to 70 ◦ C; the process continued for 2–3 h until an intense red-colored solution was obtained. The PST-Gold nanoparticles thus obtained were characterized by size with transmission electron microscopy (TEM) at an accelerated voltage of 80 kV (Hitachi TEM system). Hydrodynamic diameter and zeta potential of the nanoparticles in solution was determined by dynamic light scattering (DLS) with the help of a Zetasizer 3000 (Malvern Instruments, UK) using an argon ion laser beam at a wavelength of 488 nm and a scattering angle of 90◦ . The sample was filtered through a 0.45 m filter directly into a clean 10 mm-diameter cell. 2.2. Cell lines The murine lymphoid cancer cell lines, Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC) were procured from Amala Cancer Research Centre, Thrissur, India. DLA and EAC cells were maintained by intraperitoneal serial transplantation at a
concentration of 1 × 106 cells/mouse. For in vitro assays the cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C and 5% CO2 incubator (Heraeus BB 15). 2.3. In vitro cytotoxicity assay The growth inhibitory capacity of PST-Gold nanoparticles was evaluated in cancer cell lines using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium (MTT) assay as previously described [8,11,21]. The absorbance was measured at 570 nm using a microplate spectrophotometer (BioTek, Power Wave XS). The proliferation rate and inhibitory rate of the cells were calculated with the following formula: Proliferation rate (PR) % =
Absorbance of sample Absorbance of control
× 100
Inhibitory rate (IR) % = 100 − PR MTT assays were performed on cancer cell lines with various concentrations of PST-Gold nanoparticles ranging from 0.001 ng/ml to 1000 g/ml over a period of 24–48 h. 2.4. Acridine orange-ethidium bromide staining assay Acridine orange-ethidium bromide dual staining detects apoptosis in vitro based on the differential uptake of two fluorescent DNA binding dyes by viable and nonviable cells [23]. The experiment was performed as described earlier [11,21]. The cells were observed under an inverted fluorescent microscope under a FITC filter (Olympus 1X51, Singapore). 2.5. Antitumor activity against ascites tumors in vivo Female BALB/c mice were maintained in well-ventilated cages with free access to normal mouse food and water provided ad libitum. Temperature (25 ± 2 ◦ C) and humidity (50 ± 5%) was regulated and the illumination cycle was set to 12 h light–dark. Animal protocols were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. For the inoculation of ascites tumors in the mice, DLA or EAC cells collected from the donor mouse were suspended briefly in sterile isotonic saline. Viable cells were counted (Trypan blue assay) and adjusted to a concentration ensuring that each animal receives an intraperitoneal (i.p.) injection of 1 × 106 cells/100 l. Briefly, mice were divided into four groups; each group had three treatment protocols–PBS (vehicle), PST001 (100 mg/kg) and PST-Gold nanoparticles (100 mg/kg) under subgroups (n = 12/sub group) as summarized in Fig. 1. Both the vehicle and the compounds were administered by i.p. injection. Groups I–III were injected with DLA or EAC cells on day 1, while group IV mice were injected with cells on day 8. The compounds were administered on day 2 (group I), days 2–15 (group II), days 9–22 (group III) and days 1–7 (group IV). One half the number in each group were sacrificed on day 16 (group I and II) and day 23 (group III and IV) to determine the tumor volume, while the other half were retained for the survival analysis. To estimate the tumor volume, mice were euthanized by cervical dislocation and the abdomen was incised over a clean vessel, and the volume of the ascites fluid obtained (V1 ) noted. The peritoneal cavity was then washed with isotonic saline until the return was clear, and the volume of the saline (V2 ) was recorded. The volume of ascites tumor (V3 ) was calculated using the formula: V3 = (V1 + V2 ) − V2 . The mean survival time (MST) and percentage of increase in life span (% ILS) was calculated as previously reported [24,25]. MST = (A + B)/2, where A is the day of the first death and B is
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Fig. 1. Schematic for DLA and EAC ascites tumor models. Female mice were intraperitoneally transplanted with DLA or EAC ascites tumors and divided into four major groups–group I– IV, depending on the treatment schedule. Each of these groups have three treatment subgroups (n = 6/subgroup)–control, PST001 and PST-Gold. Compounds were administered on different days as shown. Mice were sacrificed to estimate antitumor parameters on day 16 (group I–II) or day 23 (group III–IV).
the day of last death. The percentage of increase in life span was calculated using the following formula: % ILS = (T − C)/C × 100, where T and C are the MST of treated and control animals, respectively. 2.6. Antitumor activity against EAC-induced solid-tumors in vivo EAC cells (1 × 106 cells/mouse) were injected subcutaneously with a fine needle (31G) to develop solid tumors in the hind limb of mice (n = 6/group). These experiments were conducted in a way that the compounds–PBS, PST001 and PST-Gold nanoparticles were administered by two routes, intraperitoneal (i.p.) and intratumoral (i.t.) injections, daily for 14 consecutive days, starting on day 9 after tumor inoculation (days 9–22 as shown in Fig. 1, group III). Unlike in the previous set of experiments, all the mice were used to determine tumor volume and overall survival. The radii of the developing tumors were measured every 3rd day from day 8 to day 32, using vernier calipers and the tumor volume was estimated using the formula: V = 4/3r1 2 r2 , where r1 and r2 represent the radii from two different sites [25,26]. 2.7. Statistical analysis Data represents the mean ±standard deviation (SD) of three replicates, analyzed using GraphPad PRISM v5.0 (GraphPad Software Inc., San Diego, CA). Statistically significant differences were considered if P < 0.05, as determined using one-way analysis of variance (ANOVA). SPSS 17 software (IBM, Inc., NY) was used to obtain the Kaplan–Meier survival curve. IC50 values were calculated using the Easy Plot software (Spiral Software, MD). 3. Results and discussion 3.1. Synthesis and characterization of galactoxyloglucan-gold nanoparticles The polysaccharide PST001, isolated from the seed kernels of Ti, was found to have a neutral pH, with a total sugar content of 98%, as determined by the phenol-sulfuric acid method. After isolation, the polysaccharide was purified by gel filtration chromatography, lyophilized, and stored. PST001 successfully reduced HAuCl4 to form AuNPs with a deep red color; acting itself as capping agent. Normally, a reducing agent will produce AuNPs from HAuCl4 but the newly formed nanoparticles will adhere to each other, instantly forming aggregates. In order to avoid this, a capping agent is often used which will attach to the sticky ends of the AuNPs thus preventing further aggregation. The current process
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of preparation of PST-Gold nanoparticles does not require the use of a capping agent as PST001 itself acts as a capping agent in addition to its role as a reducing agent. PST-Gold nanoparticles of different size and shape were prepared using various concentrations of PST001 utilizing the reduction and stabilization process. PST-Gold nanoparticles of an average size of 40 nm and 35 nm were prepared using 1.5 mg/ml and 5 mg/ml of PST001 respectively (Fig. 2a–d). The resultant nanoparticles were heterogeneous in morphology with few spherical nanoparticles. Homogenous and spherical PST-Gold nanoparticles of 20 nm size were prepared using 10 mg/ml PST001 (Fig. 2e,f). The zeta potential of the nanoparticles was found to be −34.6 mV (Supplementary Fig. 1). PST-Gold nanoparticles of various size and shape were evaluated for their cytotoxicity against various cancer cell lines by MTT assay. Spherical PST-Gold nanoparticles of 20 nm size were demonstrated to be a promising candidate both in terms of stability and cytotoxicity potential (data not shown). Hence, the 20 nm PST-Gold nanoparticles were selected for further biological analysis. Moreover, the high zeta potential value of the nanoparticle indicates its high stability. The preparation of gold nanoconjugates with galactoxyloglucan, PST001 for the reduction and stabilization of AuNPs does not use any hazardous chemicals or environmental toxins and is, therefore, a “green” nanoparticle synthesis process. These 20 nm PST-Gold nanoparticles are stable up to 1 year with a wide range of pH tolerance [21]. Hence, they are more stable than the commercially available borohydrate or citrate-reduced AuNPs [27]. 3.2. PST-Gold nanoparticles exert anticancer effects through induction of apoptosis The polysaccharide PST001 was reported to be an anticancer agent both in vitro and in vivo with immunomodulatory properties and, therefore, PST-Gold nanoparticles were evaluated for their cytotoxicity against murine ascites cancer cell lines DLA and EAC by MTT assay. The cytotoxic potential was found to be highly significant (P < 0.001) in all cell lines examined. DLA and EAC cells were growth-arrested with IC50 values of 31.6 ± 1.6 g/ml and 55 ± 2.1 g/ml, respectively, after 24 h of incubation with PST-Gold nanoparticles (Fig. 3a,b). The native polysaccharide PST001 produced IC50 values of 43 ± 1.3 g/ml and 597 ± 1.9 g/ml on DLA and EAC cells only after prolonged (48 h) incubation (Supplementary Table 7). Standard citrate-capped AuNPs of 20 nm in size and 1 mM HAuCl4 were found to be non-toxic to the cell lines. In order to determine the mechanism of cell death induction by PST-Gold nanoparticles, apoptotic assays were conducted after the administration of 10 g/ml of AuNPs for 24 h. Examination of AuNPtreated cells for apoptosis, using acridine orange-ethidium bromide staining showed a change in color from green to yellow/red with associated apoptotic features such as the presence of apoptotic bodies and nuclear condensation. Significant changes in fluorescence were observed for both DLA (Fig. 3c,d) and EAC cells (Fig. 3e,f) when treated with PST-Gold nanoparticles. Morphological phase contrast microscopy evaluation of cells treated with PST-Gold nanoparticles (10 g/ml) for 24 h revealed a decrease in cell number which was accompanied by salient morphological features of apoptosis, such as distorted shape, membrane blebbing, and the presence of apoptotic bodies compared to the vehicle in DLA (Fig. 3g,h) and EAC cells (Fig. 3i,j). Various polysaccharides were previously reported to have both anticancer and immunomodulatory effects [6,8,14]. However, the present study revealed that the PST-Gold nanoparticles hold more potential than other compounds. Apoptosis is the most appreciable mode of cell death, and several polysaccharides and nanoparticles have been shown to induce apoptosis in cancer cell lines. Membrane blebbing is one of the hallmarks of apoptosis which refers to the irregular bulges in the plasma membrane of the cell caused by localized decoupling of the cytoskeleton from the
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Fig. 2. TEM images and DLS spectra of various combinations of PST001 in PST-Gold nanoparticles. a, c, and e represents the TEM images of 1.5 (40 nm), 5 (35 nm) and 10 (20 nm) mg/ml of PST001 while b, d, and e represents the corresponding DLS spectra of the PST-Gold nanoparticles.
plasma membrane. The budding phenomenon of apoptotic cells lasts for only a few minutes, but the formation of the apoptotic bodies remains visible for 1–2 h [28]. The inhibition of cell proliferation exhibited by the PST-Gold nanoparticles was confirmed through the induction of apoptosis. We previously reported that the PST-Gold nanoparticles exhibited excellent anticancer potential through the induction of apoptosis in a variety of cancer cell lines such as MCF7, K562, A549, A375, HepG2, and HCT116 with specific immunomodulatory activity in vivo [21]. Even though this multimodal therapeutic efficacy by the galactoxyloglucan-derived gold nanoconjugates could be a novel approach to achieve anticancer synergy, further testing in vivo was required to establish its therapeutic utility. 3.3. Effect of PST-Gold nanoparticles on ascites tumor-bearing mice DLA and EAC ascites tumor-bearing mice were evaluated on the 16th and 23rd day of compound administration for the effects
on body weight, tumor volume, viable and non-viable tumor cell counts, and % ILS. In the DLA mice, there was a significant reduction in the tumor volume (P < 0.001) in all groups administered PST001 and PST-Gold nanoparticles, with the exception of group I where the differences in tumor volume reduction were non-significant (Fig. 4a; Supplementary Tables 1–4). In DLA-bearing mice, the best response was observed in group IV where PST-Gold nanoparticles significantly reduced the tumor volume in comparison to the control group. PST001 also showed a significant response in group IV, compared with other groups, but to a lesser extent than AuNPs. For DLA-bearing tumor mice, a maximum ILS of 96 ± 1.2% was observed with the PST-Gold nanoparticles administration in group IV (Fig. 4b; Supplementary Tables 1–4). PST001 showed the best response in group IV with ILS of 56 ± 1.1%. The Kaplan–Meier survival curves of DLA mice treated with PST001 or PST-Gold nanoparticles in different groups are shown in Fig. 4c–e. Statistical significances were achieved at P < 0.001 vs. all groups. A similar pattern was observed in EAC tumor-bearing mice with a significant reduction in tumor volume (P < 0.001) in groups
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Fig. 3. Evaluation of cytotoxicity and apoptosis in PST-Gold nanoparticles treated DLA and EAC cells. Inhibition rates are determined by MTT assay at 24 h for (a) DLA and (b) EAC cells. Results are expressed as the mean ±SD. Representative fluorescent images of apoptosis evaluation of cell lines treated with 10 g/ml PST-Gold nanoparticles for 24 h by Acridine Orange- Ethidium Bromide staining in (c) DLA control and (d) DLA-treated with PST-Gold nanoparticles; (e) EAC control and (f) EAC-treated with PST-Gold nanoparticles. Phase contrast microscopic images are shown in (g) DLA control and (h) DLA-treated with PST-Gold nanoparticles; (i) EAC control and (j) EAC-treated with PST-Gold nanoparticles.
II–IV for both the compounds, except for group I, where tumor volume parameters were non-significant for the PST001 treated group (P < 0.05), but significant for PST-Gold (Fig. 5a; Supplementary Tables 1–4). In both the compounds, even though group IV showed better tumor reduction, this difference was not significant with that of group II. There was a significant reduction in the viable tumor cell counts (P < 0.001) for DLA and EAC tumors in all groups administered compounds (Supplementary Tables 1–4). Group II also showed a significant reduction in tumor volume (P < 0.001) which was almost comparable with group IV in DLA and EAC cells. The % ILS increased significantly (P < 0.001) in
all groups. As observed in DLA mice, EAC-bearing mice treated with PST-Gold nanoparticles in group IV had a maximum ILS of 108 ± 0.9%. It is notable that in group IV PST001 showed the best effect with an increased life span of 54 ± 0.9% (Fig. 5b; Supplementary Tables 1–4). The Kaplan–Meier survival curves of EAC groups are shown in Fig. 5c–e, respectively (P < 0.001 vs. all groups). Administration of PST-Gold nanoparticles was found to be safer in animals with no noticeable side effects during the tumor regression experiments in both the ascites tumor models of DLA (Supplementary Fig. 2) and EAC (Supplementary Fig. 3).
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Fig. 4. Antitumor effects of PST001 and PST-Gold nanoparticles in DLA ascites mice. (a) Ascites tumor volume measurements between treatment groups at the end of experimental period (b) Increment in life span. Results are expressed as the mean ±SD. Statistically significant differences at *P < 0.05, ** P < 0.01, *** P < 0.001 and ns, nonsignificant, compared with the control group. Kaplan-Meier survival curve of DLA tumor bearing mice treated with PST001 and PST-Gold nanoparticles in (c) group II, (d) group III and (e) group IV.
3.4. Effect of PST-Gold nanoparticles on solid tumor-bearing mice We next evaluated the antitumor activity of PST-Gold nanoparticles in a syngeneic EAC-induced solid tumor mouse allograft. As the results suggest there was a significant reduction in the tumor burden from day 14, irrespective of the routes of drug administration. Both the i.p. (Fig. 6a, Supplementary Table 5) and i.t (Fig. 6b, Supplementary Table 6) routes showed significant solid tumor regression (P < 0.001) on treatment with PST-Gold nanoparticles and PST001. On i.p. administration, the mice treated with PST-Gold nanoparticles showed an ILS of 65 ± 2.3%, while PST001 yielded only 37.5 ± 2%. In the case of i.t. administration, PST-Gold
nanoparticles and PST001 demonstrated a substantial increment of 94.7 ± 2.3% and 42 ± 2.1% respectively (Fig. 6c). The Kaplan–Meier survival curves for i.p. and i.t. modes of compound administration for EAC solid tumor-bearing mice are shown in Fig. 6d,e. As expected in the control group, the size of solid tumor increased as the days progressed. From our results, it is quite clear that out of the two drugs – parental and gold conjugated nanoparticle derivative – the PST-Gold nanoparticles yielded the most significant solid tumor regression and increased life span on i.t. administration as compared to i.p administration. However, it should be noted that there were no differences in the trends of tumor reduction between these two drugs (Fig. 6a vs. b) on either mode of drug administration.
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Fig. 5. Antitumor effects of PST001 and PST-Gold nanoparticles in EAC ascites mice. (a) Ascites tumor volume measurements between treatment groups at the end of experimental period (b) Increment in life span. Results are expressed as the mean ±SD. Statistically significant differences at *P < 0.05, ** P < 0.01, *** P < 0.001 and ns, nonsignificant, compared with the control group. Kaplan-Meier survival curve of EAC bearing mice treated with PST001 and PST-Gold nanoparticles in (c) group II, (d) group III and (e) group IV.
Gold nanoparticles are excellent agents for therapeutic purposes due to their ease of synthesis and their ability to easily adapt to multifunctional ligands for targeted delivery [29]. Their physico-chemical aspects broaden the applications in the photodynamic and thermal ablation therapies and contrast imaging purposes. Gold nanoparticles are ideally the best for easy cancer detection and there are vast array of multifaceted gold nanoparticle systems in the preclinical phases [29]. Our study provides encouraging evidence for the antitumor effects of the compounds evaluated in ascites and solid tumor-bearing mice models. Treatment with PST-Gold nanoparticles significantly reduced the tumor volume, viable tumor cell count, and % ILS of tumor-bearing mice in both the ascites and solid tumor models. Administration of
nanoparticles via intratumoral route delivered higher response than the intraperitoneal route, but no such difference was observed in the case of PST001. This could be owing to the phenomenon of tumor leakage exhibited by the bulk molecules. Although larger macromolecules displayed the highest accumulation in tumors, they penetrated only a relatively short distance into the tumor and were mostly concentrated near the vascular surface [30]. It has been proposed that nanoparticles demonstrated increased cytotoxicity by ‘enhanced permeation and retention’ (EPR) relative to the parent polymer. The EPR effect has been demonstrated for a wide range of nanosized delivery systems, such as nanoparticles, liposomes, micelles, and dendrimers [31]. Thus nanoparticles tend to accumulate at the tumor sites more specifically than the parent polymeric
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Fig. 6. Antitumor activity of PST001 and PST-Gold nanoparticles in EAC solid carcinoma. Solid-tumor measurements were taken on days shown after (a) i.p. administration or (b) i.t. administration of compounds. Assessment of increment in life span was done in (c) EAC-induced solid-tumor bearing mice with respect to different routes of drug administration. Results are expressed as the mean ±SD. Statistically significant differences are at *** P < 0.001, as compared with the control group. Kaplan–Meier survival curve of EAC-induced solid-tumor bearing mice administered with (d) i.p. or (e) i.t. administration of PST001 and PST-Gold nanoparticles.
material, which normally excludes tumor leakage and produces a better response with less deleterious effects. A dependable standard for judging the efficiency of any anticancer drug is the prolongation of lifespan in animals. As per the NCI criteria, an ILS exceeding 25% indicates antitumor effectiveness of a drug [32], and thus the data in this study indicate that the PST-Gold nanoparticles may have superior anticancer applications compared to the parent drug in a wide variety of cancers. Although the tumor reduction exhibited by PST-Gold nanoparticles were not as high as other clinically used therapeutics, the overall survival was higher than with many known chemotherapeutics. The cause of such optimal effects without any significant toxicity may be attributable to the previously noted immunomodulatory activity [21]. Interestingly, pre-treatment of the nanoparticles also reduced the tumor volume and increased life span considerably, suggesting that it may
have an additional chemopreventive effect. Group IV was mainly targeted to demonstrate the chemopreventive activity of the compound. Chemopreventive activity of PST-Gold nanoparticles was observed to be higher in DLA than in EAC. Identifying potential tertiary chemopreventive agents to combat tumor recurrence will not only lessen the disease associated morbidity and mortality, but also indirectly reduce cancer incidence. To reiterate, for a compound to be developed further it should only have a minimal or tolerable toxicity with beneficial effects. Most chemotherapeutic agents have serious side effects which limit their widespread clinical applications, warranting the need for anticancer agents that are non-toxic to normal cells. In the current study, there were no observable side effects upon administration of the parent polysaccharide as well as its gold nanoparticle derivative, which justifies their unique drug utility. More specifically, the
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nanoconjugates maintained the safety profile of the parental polysaccharide PST001 while eliciting significant anticancer and immunomodulatory effects. Taken together, PST-Gold nanoparticles were superior to the PST001 in all the aspects of our in vivo experiments. A lowcost, naturally derived agent with no side effects, but potent antitumor activity and immense immunomodulatory potential could prove very valuable if developed into a future anticancer drug. 4. Conclusion We have demonstrated that galactoxyloglucan polysaccharides are more potent in inhibiting the proliferation and tumor growth of murine cancer cells in vitro and in vivo, especially when conjugated with gold nanoparticles. The versatile nature of the nanoparticles along with the multimodal therapeutic effects of the natural polysaccharide provides promising new platforms to cope with the much needed tumor-targeting and cell-killing activity. Better targeting efficiency with higher bioavailability and low toxicity makes galactoxyloglucan-derived gold nanoparticles a feasible drug for the prevention and therapy of cancer. Acknowledgements We greatly acknowledge the Council of Scientific and Industrial Research (CSIR), Govt. of India, for the research fellowship to the first author (MMJ); the Kerala State Council for Science, Technology and Environment (KSCSTE), Govt. of Kerala, for the financial support; and the National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, for the DLS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.12.058.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Antitumor activity of galactoxyloglucan-gold nanoparticles against murine ascites and solid carcinoma Manu M. Joseph a , S.R. Aravind a , Suraj K. George b , K. Raveendran Pillai c , S. Mini d , T.T. Sreelekha a,∗ a
Laboratory of Biopharmaceuticals and Nanomedicine, Division of Cancer Research, Regional Cancer Centre, Trivandrum, Kerala, India Dept. of Hematopathology, UT MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, 77030, TX, USA c Department of Pathology, Regional Cancer Centre, Trivandrum, Kerala, India d Department of Biochemistry, University of Kerala, Trivandrum, Kerala, India b
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 2 December 2013 Accepted 27 December 2013 Available online 9 January 2014 Keywords: Anticancer Dalton’s Lymphoma Ascites Ehrlich’s Ascites Carcinoma Polysaccharide Galactoxyloglucan Gold nanoparticle Tamarindus indica
a b s t r a c t Galactoxyloglucan polysaccharide (PST001), isolated from the seed kernels of Tamarindus indica (Ti), was used both as reducing and capping agent for the preparation of gold nanoparticles (PST-Gold) of 20 nm size. The present study evaluated the anticancer effects of the PST-Gold nanoparticles both in vitro and in vivo. The cytotoxicity was evaluated in the murine cancer cell lines, Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC). Galactoxyloglucan-gold nanoparticles (PST-Gold) not only retained the anticancer effects of PST001, but also showed enhanced cytotoxicity via induction of apoptosis even at lower doses and lesser incubation times. In vivo antitumor activity was tested in DLA and EAC murine ascites and EAC solid-tumor syngeneic mouse models. PST-Gold nanoparticles reduced tumor burden and increased median survival and life span significantly in both tumor models compared to the controls. The PST-Gold nanoparticles were very effective as a chemopreventive agent, showing the best overall response when administered prior to tumor induction. In the case of solid tumors, intratumoral administration of the PST-Gold nanoparticles yielded significant results with regard to survival and increment in lifespan as compared to intraperitoneal mode of drug administration. Further studies in higher animal models and in patients at high-risk for recurrence are warranted to fully explore and develop the potential of PST-Gold nanoconjugates as a chemopreventive and therapeutic anti-cancer agent. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a major cause of mortality worldwide and, in spite of the extraordinary effort and money spent over the past several decades, successful eradication and control remains intangible [1]. Cancer is described as a heterogeneous collection of cells that evolve in tumor microenvironments with complex ecologies [2]. The rapid progression of human cancers is related to the evasion of the body’s immune surveillance mechanisms by tumor cells through the secretion of immunosuppressive factors that modify the host immune response. Currently available chemotherapeutic modalities also suppress the immune system, predisposing cancer patients to secondary infections [3]. In this regard, there is a
∗ Corresponding author at: Scientific Officer Grade 1, Division of Cancer Research, Regional Cancer Centre (RCC), Trivandrum-11, Kerala, India. Tel.: +91 4712522378; fax: +91 4712447454. E-mail addresses: [email protected], [email protected], [email protected] (T.T. Sreelekha). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.058
significant unmet need for novel pharmaceutical agents with tumor selectivity and specificity, but with limited side effects. Tamarindus indica (Ti), a tree of the Leguminosae family, is widely grown in India and is associated with numerous beneficial effects. Previous studies have shown the predominant composition of the Ti seed kernel to be a polysaccharide, galactoxyloglucan; ˛ (1 → 4)-- D-glucan backbone substituted with side chains of d-xylopyranose and -d-galactopyranosyl (1 → 2)-␣-dxylopyranose linked (1 → 6) to glucose residues [4,5]. Antioxidant, antitumor, immunomodulatory, antimicrobial, antiulcer, and several other pharmacological activities from various polysaccharides have been reported [6–8]. Among the biopharmacological properties of polysaccharides, immunomodulatory and antitumor effects are of high priority and the majority of these agents act as biological response modifiers, which enhance immunity [9]. Ti polysaccharides have widespread utility in anticancer, immunomodulatory, antiviral, antioxidant and other pharmacological applications including its use as artificial tears in modern medicine [10–12]. They are biocompatible, biodegradable, highly viscous, and thermally stable polymers with a broad range of pH tolerance and adhesiveness [13]. Galactoxyloglucan PST001, extracted from the
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seed kernels of Ti, was previously isolated and characterized in our laboratory [10]. Earlier studies have shown that PST001 leaves normal cells unharmed, while demonstrating significant anticancer and immunomodulatory properties [10,11,14]. However, due to the advent of gold nanoparticles, a new era of combination therapeutics has begun. Gold nanoparticles (AuNPs) have the advantage of delivering drugs specifically onto the target sites [15] with unique features, including tunable surface characteristics and microenvironment stability, along with negligible side effects. AuNPs accumulate in tumor specific sites which could be utilized to trace the path of cancer cells [16]. Even though the conventional methods for the synthesis of AuNPs involve secreting toxic chemicals to the environment, the ‘green chemistry’ approach using natural products have been reported to be devoid of significant biological hazards [17,18]. Among many other natural products with potential, polysaccharides represent excellent candidates for AuNP synthesis due to the presence of hydroxyl groups, a hemiacetal reducing end and other functionalities which could play important roles in the reduction and stabilization of AuNPs. Various polysaccharides such as chitosan, cellulose, sucrose and starch have been used for the synthesis and stabilization of AuNPs [19,20]. Preliminary studies have shown that the PST-Gold nanoconjugates exert significant in vitro cytotoxicity and in vivo immunostimulatory effects with scant toxic effects in normal tissues compared to their parental counterparts [21]. However, in vivo anticancer activities in more challenging ascites solid tumor models were not investigated, which is critical for the advancement of drug to clinical trials. Thus, herein, we examined the potential of PST-Gold nanoconjugates by which the immune system of the host could be modulated to enhance the anticancer effects in vitro and in vivo. Our data show that the nanoconjugates possess superior in vitro and in vivo cytotoxicity and immunomodulatory activities in both DLA and EAC tumors. 2. Materials and methods 2.1. Preparation and characterization of PST-Gold nanoparticles Ti seeds were obtained from reliable sources and was used to isolate and characterize PST001 in the first phase following the protocol previously reported [10]. The carbohydrate content was estimated by the standard Dubois’s method [22] utilizing d-glucose as the standard. In the second phase, PST-Gold nanoparticles were prepared and characterized as described previously [21]. In brief, 3 ml of 10 mg/ml solution of PST001 was added drop-wise to 1 ml solution of HAuCl4 (1 mM) with constant stirring on a magnetic stirrer; this was then placed in a hot plate heated to 70 ◦ C; the process continued for 2–3 h until an intense red-colored solution was obtained. The PST-Gold nanoparticles thus obtained were characterized by size with transmission electron microscopy (TEM) at an accelerated voltage of 80 kV (Hitachi TEM system). Hydrodynamic diameter and zeta potential of the nanoparticles in solution was determined by dynamic light scattering (DLS) with the help of a Zetasizer 3000 (Malvern Instruments, UK) using an argon ion laser beam at a wavelength of 488 nm and a scattering angle of 90◦ . The sample was filtered through a 0.45 m filter directly into a clean 10 mm-diameter cell. 2.2. Cell lines The murine lymphoid cancer cell lines, Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC) were procured from Amala Cancer Research Centre, Thrissur, India. DLA and EAC cells were maintained by intraperitoneal serial transplantation at a
concentration of 1 × 106 cells/mouse. For in vitro assays the cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C and 5% CO2 incubator (Heraeus BB 15). 2.3. In vitro cytotoxicity assay The growth inhibitory capacity of PST-Gold nanoparticles was evaluated in cancer cell lines using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium (MTT) assay as previously described [8,11,21]. The absorbance was measured at 570 nm using a microplate spectrophotometer (BioTek, Power Wave XS). The proliferation rate and inhibitory rate of the cells were calculated with the following formula: Proliferation rate (PR) % =
Absorbance of sample Absorbance of control
× 100
Inhibitory rate (IR) % = 100 − PR MTT assays were performed on cancer cell lines with various concentrations of PST-Gold nanoparticles ranging from 0.001 ng/ml to 1000 g/ml over a period of 24–48 h. 2.4. Acridine orange-ethidium bromide staining assay Acridine orange-ethidium bromide dual staining detects apoptosis in vitro based on the differential uptake of two fluorescent DNA binding dyes by viable and nonviable cells [23]. The experiment was performed as described earlier [11,21]. The cells were observed under an inverted fluorescent microscope under a FITC filter (Olympus 1X51, Singapore). 2.5. Antitumor activity against ascites tumors in vivo Female BALB/c mice were maintained in well-ventilated cages with free access to normal mouse food and water provided ad libitum. Temperature (25 ± 2 ◦ C) and humidity (50 ± 5%) was regulated and the illumination cycle was set to 12 h light–dark. Animal protocols were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. For the inoculation of ascites tumors in the mice, DLA or EAC cells collected from the donor mouse were suspended briefly in sterile isotonic saline. Viable cells were counted (Trypan blue assay) and adjusted to a concentration ensuring that each animal receives an intraperitoneal (i.p.) injection of 1 × 106 cells/100 l. Briefly, mice were divided into four groups; each group had three treatment protocols–PBS (vehicle), PST001 (100 mg/kg) and PST-Gold nanoparticles (100 mg/kg) under subgroups (n = 12/sub group) as summarized in Fig. 1. Both the vehicle and the compounds were administered by i.p. injection. Groups I–III were injected with DLA or EAC cells on day 1, while group IV mice were injected with cells on day 8. The compounds were administered on day 2 (group I), days 2–15 (group II), days 9–22 (group III) and days 1–7 (group IV). One half the number in each group were sacrificed on day 16 (group I and II) and day 23 (group III and IV) to determine the tumor volume, while the other half were retained for the survival analysis. To estimate the tumor volume, mice were euthanized by cervical dislocation and the abdomen was incised over a clean vessel, and the volume of the ascites fluid obtained (V1 ) noted. The peritoneal cavity was then washed with isotonic saline until the return was clear, and the volume of the saline (V2 ) was recorded. The volume of ascites tumor (V3 ) was calculated using the formula: V3 = (V1 + V2 ) − V2 . The mean survival time (MST) and percentage of increase in life span (% ILS) was calculated as previously reported [24,25]. MST = (A + B)/2, where A is the day of the first death and B is
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Fig. 1. Schematic for DLA and EAC ascites tumor models. Female mice were intraperitoneally transplanted with DLA or EAC ascites tumors and divided into four major groups–group I– IV, depending on the treatment schedule. Each of these groups have three treatment subgroups (n = 6/subgroup)–control, PST001 and PST-Gold. Compounds were administered on different days as shown. Mice were sacrificed to estimate antitumor parameters on day 16 (group I–II) or day 23 (group III–IV).
the day of last death. The percentage of increase in life span was calculated using the following formula: % ILS = (T − C)/C × 100, where T and C are the MST of treated and control animals, respectively. 2.6. Antitumor activity against EAC-induced solid-tumors in vivo EAC cells (1 × 106 cells/mouse) were injected subcutaneously with a fine needle (31G) to develop solid tumors in the hind limb of mice (n = 6/group). These experiments were conducted in a way that the compounds–PBS, PST001 and PST-Gold nanoparticles were administered by two routes, intraperitoneal (i.p.) and intratumoral (i.t.) injections, daily for 14 consecutive days, starting on day 9 after tumor inoculation (days 9–22 as shown in Fig. 1, group III). Unlike in the previous set of experiments, all the mice were used to determine tumor volume and overall survival. The radii of the developing tumors were measured every 3rd day from day 8 to day 32, using vernier calipers and the tumor volume was estimated using the formula: V = 4/3r1 2 r2 , where r1 and r2 represent the radii from two different sites [25,26]. 2.7. Statistical analysis Data represents the mean ±standard deviation (SD) of three replicates, analyzed using GraphPad PRISM v5.0 (GraphPad Software Inc., San Diego, CA). Statistically significant differences were considered if P < 0.05, as determined using one-way analysis of variance (ANOVA). SPSS 17 software (IBM, Inc., NY) was used to obtain the Kaplan–Meier survival curve. IC50 values were calculated using the Easy Plot software (Spiral Software, MD). 3. Results and discussion 3.1. Synthesis and characterization of galactoxyloglucan-gold nanoparticles The polysaccharide PST001, isolated from the seed kernels of Ti, was found to have a neutral pH, with a total sugar content of 98%, as determined by the phenol-sulfuric acid method. After isolation, the polysaccharide was purified by gel filtration chromatography, lyophilized, and stored. PST001 successfully reduced HAuCl4 to form AuNPs with a deep red color; acting itself as capping agent. Normally, a reducing agent will produce AuNPs from HAuCl4 but the newly formed nanoparticles will adhere to each other, instantly forming aggregates. In order to avoid this, a capping agent is often used which will attach to the sticky ends of the AuNPs thus preventing further aggregation. The current process
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of preparation of PST-Gold nanoparticles does not require the use of a capping agent as PST001 itself acts as a capping agent in addition to its role as a reducing agent. PST-Gold nanoparticles of different size and shape were prepared using various concentrations of PST001 utilizing the reduction and stabilization process. PST-Gold nanoparticles of an average size of 40 nm and 35 nm were prepared using 1.5 mg/ml and 5 mg/ml of PST001 respectively (Fig. 2a–d). The resultant nanoparticles were heterogeneous in morphology with few spherical nanoparticles. Homogenous and spherical PST-Gold nanoparticles of 20 nm size were prepared using 10 mg/ml PST001 (Fig. 2e,f). The zeta potential of the nanoparticles was found to be −34.6 mV (Supplementary Fig. 1). PST-Gold nanoparticles of various size and shape were evaluated for their cytotoxicity against various cancer cell lines by MTT assay. Spherical PST-Gold nanoparticles of 20 nm size were demonstrated to be a promising candidate both in terms of stability and cytotoxicity potential (data not shown). Hence, the 20 nm PST-Gold nanoparticles were selected for further biological analysis. Moreover, the high zeta potential value of the nanoparticle indicates its high stability. The preparation of gold nanoconjugates with galactoxyloglucan, PST001 for the reduction and stabilization of AuNPs does not use any hazardous chemicals or environmental toxins and is, therefore, a “green” nanoparticle synthesis process. These 20 nm PST-Gold nanoparticles are stable up to 1 year with a wide range of pH tolerance [21]. Hence, they are more stable than the commercially available borohydrate or citrate-reduced AuNPs [27]. 3.2. PST-Gold nanoparticles exert anticancer effects through induction of apoptosis The polysaccharide PST001 was reported to be an anticancer agent both in vitro and in vivo with immunomodulatory properties and, therefore, PST-Gold nanoparticles were evaluated for their cytotoxicity against murine ascites cancer cell lines DLA and EAC by MTT assay. The cytotoxic potential was found to be highly significant (P < 0.001) in all cell lines examined. DLA and EAC cells were growth-arrested with IC50 values of 31.6 ± 1.6 g/ml and 55 ± 2.1 g/ml, respectively, after 24 h of incubation with PST-Gold nanoparticles (Fig. 3a,b). The native polysaccharide PST001 produced IC50 values of 43 ± 1.3 g/ml and 597 ± 1.9 g/ml on DLA and EAC cells only after prolonged (48 h) incubation (Supplementary Table 7). Standard citrate-capped AuNPs of 20 nm in size and 1 mM HAuCl4 were found to be non-toxic to the cell lines. In order to determine the mechanism of cell death induction by PST-Gold nanoparticles, apoptotic assays were conducted after the administration of 10 g/ml of AuNPs for 24 h. Examination of AuNPtreated cells for apoptosis, using acridine orange-ethidium bromide staining showed a change in color from green to yellow/red with associated apoptotic features such as the presence of apoptotic bodies and nuclear condensation. Significant changes in fluorescence were observed for both DLA (Fig. 3c,d) and EAC cells (Fig. 3e,f) when treated with PST-Gold nanoparticles. Morphological phase contrast microscopy evaluation of cells treated with PST-Gold nanoparticles (10 g/ml) for 24 h revealed a decrease in cell number which was accompanied by salient morphological features of apoptosis, such as distorted shape, membrane blebbing, and the presence of apoptotic bodies compared to the vehicle in DLA (Fig. 3g,h) and EAC cells (Fig. 3i,j). Various polysaccharides were previously reported to have both anticancer and immunomodulatory effects [6,8,14]. However, the present study revealed that the PST-Gold nanoparticles hold more potential than other compounds. Apoptosis is the most appreciable mode of cell death, and several polysaccharides and nanoparticles have been shown to induce apoptosis in cancer cell lines. Membrane blebbing is one of the hallmarks of apoptosis which refers to the irregular bulges in the plasma membrane of the cell caused by localized decoupling of the cytoskeleton from the
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Fig. 2. TEM images and DLS spectra of various combinations of PST001 in PST-Gold nanoparticles. a, c, and e represents the TEM images of 1.5 (40 nm), 5 (35 nm) and 10 (20 nm) mg/ml of PST001 while b, d, and e represents the corresponding DLS spectra of the PST-Gold nanoparticles.
plasma membrane. The budding phenomenon of apoptotic cells lasts for only a few minutes, but the formation of the apoptotic bodies remains visible for 1–2 h [28]. The inhibition of cell proliferation exhibited by the PST-Gold nanoparticles was confirmed through the induction of apoptosis. We previously reported that the PST-Gold nanoparticles exhibited excellent anticancer potential through the induction of apoptosis in a variety of cancer cell lines such as MCF7, K562, A549, A375, HepG2, and HCT116 with specific immunomodulatory activity in vivo [21]. Even though this multimodal therapeutic efficacy by the galactoxyloglucan-derived gold nanoconjugates could be a novel approach to achieve anticancer synergy, further testing in vivo was required to establish its therapeutic utility. 3.3. Effect of PST-Gold nanoparticles on ascites tumor-bearing mice DLA and EAC ascites tumor-bearing mice were evaluated on the 16th and 23rd day of compound administration for the effects
on body weight, tumor volume, viable and non-viable tumor cell counts, and % ILS. In the DLA mice, there was a significant reduction in the tumor volume (P < 0.001) in all groups administered PST001 and PST-Gold nanoparticles, with the exception of group I where the differences in tumor volume reduction were non-significant (Fig. 4a; Supplementary Tables 1–4). In DLA-bearing mice, the best response was observed in group IV where PST-Gold nanoparticles significantly reduced the tumor volume in comparison to the control group. PST001 also showed a significant response in group IV, compared with other groups, but to a lesser extent than AuNPs. For DLA-bearing tumor mice, a maximum ILS of 96 ± 1.2% was observed with the PST-Gold nanoparticles administration in group IV (Fig. 4b; Supplementary Tables 1–4). PST001 showed the best response in group IV with ILS of 56 ± 1.1%. The Kaplan–Meier survival curves of DLA mice treated with PST001 or PST-Gold nanoparticles in different groups are shown in Fig. 4c–e. Statistical significances were achieved at P < 0.001 vs. all groups. A similar pattern was observed in EAC tumor-bearing mice with a significant reduction in tumor volume (P < 0.001) in groups
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Fig. 3. Evaluation of cytotoxicity and apoptosis in PST-Gold nanoparticles treated DLA and EAC cells. Inhibition rates are determined by MTT assay at 24 h for (a) DLA and (b) EAC cells. Results are expressed as the mean ±SD. Representative fluorescent images of apoptosis evaluation of cell lines treated with 10 g/ml PST-Gold nanoparticles for 24 h by Acridine Orange- Ethidium Bromide staining in (c) DLA control and (d) DLA-treated with PST-Gold nanoparticles; (e) EAC control and (f) EAC-treated with PST-Gold nanoparticles. Phase contrast microscopic images are shown in (g) DLA control and (h) DLA-treated with PST-Gold nanoparticles; (i) EAC control and (j) EAC-treated with PST-Gold nanoparticles.
II–IV for both the compounds, except for group I, where tumor volume parameters were non-significant for the PST001 treated group (P < 0.05), but significant for PST-Gold (Fig. 5a; Supplementary Tables 1–4). In both the compounds, even though group IV showed better tumor reduction, this difference was not significant with that of group II. There was a significant reduction in the viable tumor cell counts (P < 0.001) for DLA and EAC tumors in all groups administered compounds (Supplementary Tables 1–4). Group II also showed a significant reduction in tumor volume (P < 0.001) which was almost comparable with group IV in DLA and EAC cells. The % ILS increased significantly (P < 0.001) in
all groups. As observed in DLA mice, EAC-bearing mice treated with PST-Gold nanoparticles in group IV had a maximum ILS of 108 ± 0.9%. It is notable that in group IV PST001 showed the best effect with an increased life span of 54 ± 0.9% (Fig. 5b; Supplementary Tables 1–4). The Kaplan–Meier survival curves of EAC groups are shown in Fig. 5c–e, respectively (P < 0.001 vs. all groups). Administration of PST-Gold nanoparticles was found to be safer in animals with no noticeable side effects during the tumor regression experiments in both the ascites tumor models of DLA (Supplementary Fig. 2) and EAC (Supplementary Fig. 3).
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Fig. 4. Antitumor effects of PST001 and PST-Gold nanoparticles in DLA ascites mice. (a) Ascites tumor volume measurements between treatment groups at the end of experimental period (b) Increment in life span. Results are expressed as the mean ±SD. Statistically significant differences at *P < 0.05, ** P < 0.01, *** P < 0.001 and ns, nonsignificant, compared with the control group. Kaplan-Meier survival curve of DLA tumor bearing mice treated with PST001 and PST-Gold nanoparticles in (c) group II, (d) group III and (e) group IV.
3.4. Effect of PST-Gold nanoparticles on solid tumor-bearing mice We next evaluated the antitumor activity of PST-Gold nanoparticles in a syngeneic EAC-induced solid tumor mouse allograft. As the results suggest there was a significant reduction in the tumor burden from day 14, irrespective of the routes of drug administration. Both the i.p. (Fig. 6a, Supplementary Table 5) and i.t (Fig. 6b, Supplementary Table 6) routes showed significant solid tumor regression (P < 0.001) on treatment with PST-Gold nanoparticles and PST001. On i.p. administration, the mice treated with PST-Gold nanoparticles showed an ILS of 65 ± 2.3%, while PST001 yielded only 37.5 ± 2%. In the case of i.t. administration, PST-Gold
nanoparticles and PST001 demonstrated a substantial increment of 94.7 ± 2.3% and 42 ± 2.1% respectively (Fig. 6c). The Kaplan–Meier survival curves for i.p. and i.t. modes of compound administration for EAC solid tumor-bearing mice are shown in Fig. 6d,e. As expected in the control group, the size of solid tumor increased as the days progressed. From our results, it is quite clear that out of the two drugs – parental and gold conjugated nanoparticle derivative – the PST-Gold nanoparticles yielded the most significant solid tumor regression and increased life span on i.t. administration as compared to i.p administration. However, it should be noted that there were no differences in the trends of tumor reduction between these two drugs (Fig. 6a vs. b) on either mode of drug administration.
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Fig. 5. Antitumor effects of PST001 and PST-Gold nanoparticles in EAC ascites mice. (a) Ascites tumor volume measurements between treatment groups at the end of experimental period (b) Increment in life span. Results are expressed as the mean ±SD. Statistically significant differences at *P < 0.05, ** P < 0.01, *** P < 0.001 and ns, nonsignificant, compared with the control group. Kaplan-Meier survival curve of EAC bearing mice treated with PST001 and PST-Gold nanoparticles in (c) group II, (d) group III and (e) group IV.
Gold nanoparticles are excellent agents for therapeutic purposes due to their ease of synthesis and their ability to easily adapt to multifunctional ligands for targeted delivery [29]. Their physico-chemical aspects broaden the applications in the photodynamic and thermal ablation therapies and contrast imaging purposes. Gold nanoparticles are ideally the best for easy cancer detection and there are vast array of multifaceted gold nanoparticle systems in the preclinical phases [29]. Our study provides encouraging evidence for the antitumor effects of the compounds evaluated in ascites and solid tumor-bearing mice models. Treatment with PST-Gold nanoparticles significantly reduced the tumor volume, viable tumor cell count, and % ILS of tumor-bearing mice in both the ascites and solid tumor models. Administration of
nanoparticles via intratumoral route delivered higher response than the intraperitoneal route, but no such difference was observed in the case of PST001. This could be owing to the phenomenon of tumor leakage exhibited by the bulk molecules. Although larger macromolecules displayed the highest accumulation in tumors, they penetrated only a relatively short distance into the tumor and were mostly concentrated near the vascular surface [30]. It has been proposed that nanoparticles demonstrated increased cytotoxicity by ‘enhanced permeation and retention’ (EPR) relative to the parent polymer. The EPR effect has been demonstrated for a wide range of nanosized delivery systems, such as nanoparticles, liposomes, micelles, and dendrimers [31]. Thus nanoparticles tend to accumulate at the tumor sites more specifically than the parent polymeric
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Fig. 6. Antitumor activity of PST001 and PST-Gold nanoparticles in EAC solid carcinoma. Solid-tumor measurements were taken on days shown after (a) i.p. administration or (b) i.t. administration of compounds. Assessment of increment in life span was done in (c) EAC-induced solid-tumor bearing mice with respect to different routes of drug administration. Results are expressed as the mean ±SD. Statistically significant differences are at *** P < 0.001, as compared with the control group. Kaplan–Meier survival curve of EAC-induced solid-tumor bearing mice administered with (d) i.p. or (e) i.t. administration of PST001 and PST-Gold nanoparticles.
material, which normally excludes tumor leakage and produces a better response with less deleterious effects. A dependable standard for judging the efficiency of any anticancer drug is the prolongation of lifespan in animals. As per the NCI criteria, an ILS exceeding 25% indicates antitumor effectiveness of a drug [32], and thus the data in this study indicate that the PST-Gold nanoparticles may have superior anticancer applications compared to the parent drug in a wide variety of cancers. Although the tumor reduction exhibited by PST-Gold nanoparticles were not as high as other clinically used therapeutics, the overall survival was higher than with many known chemotherapeutics. The cause of such optimal effects without any significant toxicity may be attributable to the previously noted immunomodulatory activity [21]. Interestingly, pre-treatment of the nanoparticles also reduced the tumor volume and increased life span considerably, suggesting that it may
have an additional chemopreventive effect. Group IV was mainly targeted to demonstrate the chemopreventive activity of the compound. Chemopreventive activity of PST-Gold nanoparticles was observed to be higher in DLA than in EAC. Identifying potential tertiary chemopreventive agents to combat tumor recurrence will not only lessen the disease associated morbidity and mortality, but also indirectly reduce cancer incidence. To reiterate, for a compound to be developed further it should only have a minimal or tolerable toxicity with beneficial effects. Most chemotherapeutic agents have serious side effects which limit their widespread clinical applications, warranting the need for anticancer agents that are non-toxic to normal cells. In the current study, there were no observable side effects upon administration of the parent polysaccharide as well as its gold nanoparticle derivative, which justifies their unique drug utility. More specifically, the
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nanoconjugates maintained the safety profile of the parental polysaccharide PST001 while eliciting significant anticancer and immunomodulatory effects. Taken together, PST-Gold nanoparticles were superior to the PST001 in all the aspects of our in vivo experiments. A lowcost, naturally derived agent with no side effects, but potent antitumor activity and immense immunomodulatory potential could prove very valuable if developed into a future anticancer drug. 4. Conclusion We have demonstrated that galactoxyloglucan polysaccharides are more potent in inhibiting the proliferation and tumor growth of murine cancer cells in vitro and in vivo, especially when conjugated with gold nanoparticles. The versatile nature of the nanoparticles along with the multimodal therapeutic effects of the natural polysaccharide provides promising new platforms to cope with the much needed tumor-targeting and cell-killing activity. Better targeting efficiency with higher bioavailability and low toxicity makes galactoxyloglucan-derived gold nanoparticles a feasible drug for the prevention and therapy of cancer. Acknowledgements We greatly acknowledge the Council of Scientific and Industrial Research (CSIR), Govt. of India, for the research fellowship to the first author (MMJ); the Kerala State Council for Science, Technology and Environment (KSCSTE), Govt. of Kerala, for the financial support; and the National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, for the DLS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.12.058.
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