1. Introduction
Colorectal cancer is the most common type of gastrointestinal cancer, accounting for over 9% of all cancer cases [
1]. According to annual reports issued by the Ministry of Health and Welfare in Taiwan, colorectal cancer is second only to breast cancer in incidence and ranks third in mortality. It is also the fourth most common cause of death throughout the world [
2]. Peritoneal metastasis is one of the phenomena in patients suffering from colorectal, gastric or ovarian cancer at the late phase [
3,
4,
5,
6]. Patients with peritoneal metastasis exhibit poor prognosis, with only a 12 month median survival, either with or without systemic chemotherapy [
7,
8].
Nanoparticles (NPs) can encapsulate poorly soluble drugs, fluorescent dyes, and imaging contrast agents and have multifunctional biological applications. In a solid tumor, the distribution of NPs is adjusted by the two main effects of passive targeting, also named enhanced permeability and retention (EPR) effect [
9], and active targeting [
10]. Based on the rapid development of nanotechnology, nanoparticle (NP)-based theranostic agents, also called nanotheranostic agents, have provided an opportunity to achieve individualized treatment [
11].
Various sizes and shapes of gold nanoparticles (AuNPs) with different physical and chemical properties have been developed and have led to an expansion in medical applications [
12,
13]. For example, gold nanoclusters have been used as a fluorescent dye [
14], and gold nanospheres have been employed as contrast agent in computed tomography (CT) for cancer diagnosis [
15,
16]. Gold nanospheres could be applied as a radiosensitizer [
17]. Gold nanoshells and gold nanorods exhibit distinctive surface plasmon resonance and are widely applied in photothermal therapy [
18,
19,
20].
Albumin, with its non-antigenicity, biocompatibility, high protein binding of various drugs (such as paclitaxel [
21], doxorubicin [
22], and lapatinib [
23]), and biodegradability, is regarded as an ideal material in drug delivery devices [
24,
25]. In addition, some albumin-binding proteins, e.g., glycoprotein gp60 and secreted protein acidic and rich in cysteine, have been found to be overexpressed in breast cancer, non-small-cell lung cancer, and colon cancer [
26,
27]. Several albumin-conjugated drugs and albumin-based nanoparticles have been reported for cancer diagnosis and therapy [
28,
29,
30]. In 2005, Abraxane
®, a paclitaxel albumin-bound particle, was approved by the United States Food and Drug Administration for treatment of metastatic breast cancer, locally advanced or metastatic non-small lung cancer, and metastatic adenocarcinoma of the pancreas.
To combine the merits of both protein and inorganic nanocarriers, the development of hybrid protein-inorganic nanoparticles for drug delivery and cancer diagnostics has been progressively enlarged [
30,
31]. The super paramagnetic iron oxide (SPIO)-encapsulated albumin nanoparticle was developed for positron emission tomography (PET)/near-infrared fluorescence (NIRF)/magnetic resonance imaging (MRI) triple functional imaging [
32] and as a drug delivery vehicle for doxorubicin [
33]. Peralta and his co-workers reported that paclitaxel-loaded gold nanorod encapsulated human serum albumin nanoparticle exhibited superior tumor growth inhibition [
34].
In the 1970s, intraperitoneal chemotherapy (IPC), which delivers a high concentration of cytotoxic drugs through the intraperitoneal route, was first introduced for patients with malignant ascites [
35]. As the presence of a peritoneal–plasma barrier maintains a high concentration gradient of cytotoxic drugs between the peritoneal cavity and the plasma compartment, chemo drugs that are directly delivered into the peritoneal cavity show a pharmacokinetic advantage over other routes of administration. Williamson et al. reported that patients receiving a nanoparticulate formulation of paclitaxel (Nanotax
®) through ip administration exhibited a higher and prolonged paclitaxel level in the abdominal cavity than those receiving iv injection in a phase I clinical trial [
36]. Cytoreductive surgery combined IPC or hyperthermic intraperitoneal chemotherapy are two common strategies for peritoneal metastasis [
37,
38]. Intraperitoneal injections of Nab-paclitaxel in mice bearing OCUM-2MD3 peritoneal xenografts have shown superior therapeutic efficacy than those receiving iv injections of Nab-paclitaxel or ip injections of paclitaxel [
39].
Previous studies have revealed the great potency of protein-inorganic nanoparticles for various solid tumor treatments. However, usage of these nanoparticles for treating peritoneal metastases remains unclear. This study developed a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP) as a drug delivery system. Indium-111 labeled Au@HSANP (111In-Au@HSANP) was prepared as the radioactive surrogate of Au@HSANP and used to investigate the biological behaviors in a CT-26 tumor/ascites mouse model after different routes of administration.
3. Discussion
Previous studies have demonstrated that opsonization followed by macrophage and Kupffer cells uptake accounts for the high MPS accumulation of most nanoparticles after iv administration [
40,
41,
42,
43]. The high level in MPS is the most significant known limitation of nanoparticulate drug delivery system. The circulation time, tumor targeting, and microdistribution in tumor regions are dependent on several factors such as the shape, size, and surface characteristics of NPs. A size less than 10 nm could avoid clearance by first pass renal filtration [
44]. The blood clearance rate of particles with diameters <200 nm is slower than that for particles with diameters over 200 nm [
45]. In this study, most radioactivity accumulated in MPS was noticed after iv injections of
111In-Au@HSANP. The results of the pharmacokinetics and biodistribution studies revealed that iv administration of Au@HSANP with an average size of 213.3 ± 32.9 nm exhibited a short distribution half-life (T
1/2α = 0.05 h) and a much longer elimination half-life (T
1/2β = 19.7 h). The radioactivity accumulation in the liver and spleen were around 16 and 40%ID/g at one hour p.i. Kinoshita et al. reported that nearly 20%ID/g of Abraxane
® (130 nm) accumulated in the liver at one hour post iv injection, while less than 1%ID/g accumulates in tumor lesions in CT-26 tumor-bearing nu/nu mice [
46]. In 2015, Qi et al. demonstrated high liver accumulation (19.3%ID/g) of Doxorubicin-loaded HSANPs (170 nm) at one hour post iv administration [
22]. Previous studies have reported that the accumulation of albumin or drugs in the lung is remarkably soon (within one hour) after iv injection of albumin [
47], albumin-conjugated drugs [
28], or albumin-based nanoparticles [
22]. The expression of gp60 in lung microvascular endothelial cells [
48] and the filtration effect of the lung capillary bed [
28] may account for the noticeable lung accumulation. This study also noticed a significant radioactivity accumulation in the lung (13.31 ± 6.49%ID/g) at one hour p.i., which then declined to 1.68 ± 0.85%ID/g at 24 h p.i.
The results of the biodistribution study showed prolonged
111In-Au@HSANP retention in the peritoneal cavity after ip administration. The area under the radioactivity-time curves (AUCs) of the critical organs derived from the biodistribution study are summarized in
Table 5. Compared with the organ AUC after iv injection, the liver and splenic AUC after ip injection were much less (about 12 and 11-fold lower). Benefitting from the low uptake in the MPS organs, the tumors and ascites in the ip injection group exhibited a 20- and 93-fold higher accumulation, respectively, than those receiving iv injections. Unlike the MPS organs, the kidneys, which are the critical excretion organ of
111In-Au@HSANP, displayed a similar radioactivity distribution profile in both the iv and ip-injection mice. The AUCs of kidney in iv-injection group was only 1.7-fold higher than that of ip-injection group. The lower uptake in the kidneys at one-hour post ip injection could be attributed to less
111In-Au@HSANP being absorbed from the peritoneum. The accumulation of
111In-Au@HSANP was 3.55 ± 1.00%ID/g at one-hour iv p.i., while only 0.41 ± 0.06%ID/g accumulated in the ip-injected group. These results revealed that the administration route of Au@HSANP played an important role in biodistribution but not in excretion.
The physicochemical properties of drugs, such as hydrophilicity, molecular weight, and particle size, influence, absorption in the peritoneal cavity [
49,
50]. Drugs with a small molecular weight exhibit a shorter residence time in the peritoneal cavity [
51]. Normally, the half-life of small molecular weight drugs such as docetaxel and paclitaxel is less than 24 h after ip administration [
51,
52]. Drugs and NPs in the peritoneal cavity are absorbed by the capillaries and transferred to systemic circulation [
53]. To lengthen the retention time of intraperitoneally administrated drugs in the peritoneal cavity, several formulations and drug delivery systems, such as microparticles, micelles and liposomes have been introduced [
54]. Gelderblom et al. reported that paclitaxel entrapped in cremophor EL micelles (Taxol
®), a nonionic castor oil derivative, exhibits a pharmacokinetic advantage for peritoneal cavity exposure after ip administration, as compared to cremophor EL-free paclitaxel formulations [
55]. However, cremophor EL and ethanol-based formulations are associated with severe side effects including hypersensitivity reactions and peripheral neuropathy [
56].
The size of a drug delivery system also influences the residence time in the peritoneum after ip administration. Fujiyama et al. developed a biodegradable glycolic acid–lactic acid copolymer microsphere for incorporating cisplatin (CDDP-MS) with a mean size of 19.6 μm. After ip administration, CDDP-MS exhibits a lower acute toxicity and higher retention of cisplatin in the peritoneum as compared to cisplatin solution [
57]. Lu et al. reported that prolonged retention of paclitaxel-loaded microparticles (4 μm) could be noticed in the peritoneal cavity after ip administration [
58]. However, Kohane et al. found several peritoneal adhesions and chronic inflammation in the peritoneum after ip injections of 5–250 μm poly(lactic-co-glycolic) acid microparticles [
59]. As compared to microparticles, nanoscale particles can distribute more evenly in the peritoneal cavity and be more easily internalized by tumor cells [
60,
61]. Liposomes have been widely studied as potential carriers for hydrophilic and hydrophobic drugs and diagnostic agents [
62]. Hirano et al. demonstrated that liposomes with a smaller size (~50 nm) pass through the lymph nodes more easily than those with a larger size (~700 nm) [
63]. Studies by Dadashzadeh et al. showed that both 100 nm and 1000 nm of positively charged non-PEGylated liposomes provided greater peritoneal levels and retention. PEGylated liposome shows high peritoneal retention due to the primary tumor targeting EPR effect and also by the avoidance of macrophages present in the peritoneal cavity [
64]. In our previous study, we reported that the AUCs of ascites and tumors of
111In-labeled PEGylated liposomal vinorelbine (IVNBPL, ~100 nm) after ip administration were 6.8- and 1.7-folds higher than that of the iv-injected group [
65]. In this study, the AUC of the ascites and tumors of
111In-Au@HSANP in the same CT-26 tumor/ascites mouse model receiving ip administration were 93-fold and 20-fold higher than that received iv injection, respectively. In addition, it was found that the AUCs of
111In-Au@HSANP in the liver and spleen were 22.6 and 8.5-foldlower than that of IVNBPL, respectively. Hence, Au@HSANP could be a better drug delivery system for ip administration.
4. Materials and Methods
4.1. Materials and Reagents
Human serum albumins and glutaraldehyde solution (25% v/v) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Anhydrous ethanol (>95%) was purchased from J.T. Baker Inc. (Phillipsburg, NJ, USA). Hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O) was purchased from Alfa Aesar (Haverhill, MA, USA). Sodium citrate dehydrate was purchased from Merck (Darmstadt, Germany). S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA) was purchased from Macrocyclics (Dallas, TX, USA). The 111In-InCl3 solution was obtained from the Institute of Nuclear Energy Research (Taoyuan, Taiwan). Cell culture dishes, flasks, and plastic ware were purchased from Corning Inc (Corning, NY, USA). Fetal bovine serum and cell culture medium were purchased from HyClone (Logan, UT, USA). Sepharose 4B gel and Poly-Prep chromatography columns were purchased from GE Healthcare (Chalfont St. Giles, UK) and Bio-Rad (Hercules, CA, USA), respectively.
4.2. Preparation of Gold Nanoparticles (AuNP)
Gold nanoparticles with a diameter of 20 nm were prepared using the Turkevich’s method [
66]. All glassware and Teflon-coated magnetic bars for synthesis were cleaned using aqua regia (conc. HCl/conc. HNO
3 = 3/1,
v/
v), washed with deionized water and dried prior to use. Three hundred milliliters of deionized water (dH
2O) was heated and vigorously stirred using a Teflon-coated magnetic bar. While boiling, 3 mL of 25 mM HAuCl
4 solution and 3 mL of 50 mM trisodium citrate were sequentially added with continuous stirring for 15 min. The obtained solution was centrifuged at 6000×
g for 30 min. The pellets were washed in dH
2O three times and dispersed in the dH
2O. The UV/Vis spectrum of gold nanoparticles was recorded using a Jasco V-530 UV/VISIBLE spectrophotometer (Tokyo, Japan). The particle size and morphology were analyzed with dynamic light scattering (DLS, HORIBA, Kyoto, Japan) and transmission electron microscopy (TEM, JEOL JEM-1400plus, Tokyo, Japan), respectively. The concentration of the gold nanoparticle solution was calculated by the following formula reported by Haiss et al. [
67],
where
N is number of nanoparticles in 1 mL,
A450 is the absorbance at λ = 450 nm, and
d is the particle diameter in nm.
4.3. Preparation of AuNP-Encapsulated Human Serum Albumin Nanoparticle (Au@HSANP)
AuNP-encapsulated human serum albumin nanoparticles were prepared using the desolvation method with a slight modification [
68]. Briefly, 10 mg of human serum albumin (HSA) was dissolved in an AuNP solution (1 × 10
12 particles/mL dH
2O) and stirred at an ambient temperature for four hours. Next, 3 mL of ethanol was added dropwise (1 mL/min) and the mixture gradually became turbid. For crosslinking, 4.7 μL of 8% glutaraldehyde was added and then stirred at an ambient temperature for 24 h. The crude product Au@HSANP was purified through three cycles of centrifugation (3000×
g, 10 min) and re-dispersion in dH
2O. The final product, Au@HSANP, was kept at 4 °C before use. To investigate the stability and physiochemical properties of Au@HSANP, purified Au@HSANP was incubated in dH
2O at 4 °C. The particle size, zeta potential, polydispersity index (PDI), and morphology of Au@HSANP were determined by DLS and TEM.
4.4. Cytotoxicity of Au@HSANP in CT-26 Cell Culture
An MTT assay was used for determination of the cytotoxicity of Au@HSANP in CT-26 colon adenocarcinoma cell cultures. In brief, 5000 cells were seeded in a 96-well plate for at least eight hours before the experiment. The cells were then incubated at different concentration of Au@HSANP (0–500 ppm). At 24 and 48 h post-incubation, the cells were washed twice with PBS, and then 150 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Corp) was added and incubated for another four hours at 37 °C. Next, the medium was removed, and the formed formazan crystals were subsequently dissolved in 150 μL of dimethyl sulfoxide. The optical density at 570 nm of each well was recorded by using an ELISA reader (TECAN Trading AG, Mannedorf, Switzerland). The cell viability was calculated according the following formula.
4.5. Preparation of 111In-Labeled Au@HSANP (111In-Au@HSANP)
The preparation of radioactive Au@HSANP was based on previous study with a slight modification [
69]. Au@HSANP was conjugated with the bifunctional chelator, p-SCN-Bn-DTPA in a 0.05 M carbonate buffer (pH 8.5), at a molar ratio of 1:20 (HSA/DTPA) for two hours. The reaction mixture was centrifuged at 3000×
g for 10 min and the pellet was resuspened in dH
2O to obtain purified DTPA-Au@HSANP. The radiometal-labeling of DTPA-Au@HSANP was conducted by incubation with the appropriate amount of
111In-InCl
3 in 0.1 M citrate buffer (pH 6.0) at 37 °C for 20 min. The crude solution was subjected to gel filtration chromatography with a Sepharose 4B gel column to afford the purified
111In-Au@HSANP with a specific activity of 10–20 mCi
111In/mg Au@HSANP. The labeling efficiency and radiochemical purity of
111In-Au@HSANP were determined using instant thin layer chromatography (iTLC) with a 0.5 M sodium citrate buffer as the developing agent.
4.6. Serum Stability Assay of 111In-Au@HSANP
The serum stability of
111In-Au@HSANP was assayed on the basis of a radiochemical purity determination. An adequate radioactivity of purified
111In-Au@HSANP was incubated in normal saline (4 °C) or fetal bovine serum (FBS, 37 °C) for four, 24, 48, and 72 h. The size exclusion chromatography with a 2 mL Sepharose 4B gel column was used to determine the radiochemical purity. At least ten fractions (0.2 mL/tube) were collected and the radioactivity of each fraction was determined. The radiochemical purity was calculated by the following formula.
4.7. Cell Culture and Tumor/Ascites Animal Model
The CT-26 colon tumor/ascites-bearing mouse model was established based on a previous study [
70]. Male BALB/c mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in cages under controlled environmental conditions, with food and water being provided ad libitum. The CT-26 ascites/tumor mice model was developed, though ip injection of CT-26 cells (2 × 10
5) in a 0.5 mL FBS-free RPMI medium. Animal studies were conducted after 10–14 days inoculation. The animal experiment protocols were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University (Taipei, Taiwan, IACUC no: 1040509r).
4.8. Pharmacokinetic Study of the CT-26 Tumor/Ascites-Bearing Mouse Model after iv or ip Injections of 111In-Au@HSANP
Ten tumor/ascites-bearing mice were randomly divided into two groups. The mice received 45 μCi of 111In-Au@HSANP (200 μg of Au@HSANP) by either an iv or ip injection. One microliter of blood samples was collected from the tail vein at 0.083, 0.166, 0.75, 1, 2, 4, 8, 12, 16, 20, 24, 32, and 48 h post iv/ip administration and the radioactivity of each sample was assayed using a Wallac 1470 Wizard Gamma counter (GMI, Inc., Ramsey, MN, USA). Data were expressed as the percentage of the injected dose per milliliter (%ID/mL). The pharmacokinetic parameters were calculated using WinNonlin software (version 6.1, Pharsight Corp., Mountain View, CA, USA) using a two-compartment model for the mice receiving iv injection and a non-compartment model for those receiving ip administration.
4.9. MicroSPECT Imaging of the CT-26 Tumor/Ascites-Bearing Mouse Model after iv or ip Injection of 111In-Au@HSANP
MicroSPECT images of the CT-26 tumor/ascites-bearing mice were obtained by using a microSPECT/CT scanner (FLEX Triumph Regular FLEX X-OCT, SPECT CZT 3 Head System, Gamma Medica, Northridge, CA, USA). The mice were anesthetized with 1–2% isoflurane (w/v) in 2 L of oxygen in the supine position. MicroSPECT imaging was performed at one, four, 24, 48 and 72 h after the iv/ip injection of 111In-Au@HSANP (340–360 μCi in 0.1 mL). Images were acquired and reconstructed using an ordered-subset expectation maximization algorithm (five iterations and eight subsets).
4.10. Biodistribution of CT-26 Tumor/Ascites-Bearing Mouse Model after iv or ip Injections of 111In-Au@HSANP
Sixteen CT-26 tumor/ascites-bearing mice were randomly divided into two groups. Each mouse received 45 μCi of 111In-Au@HSANP (200 μg of Au@HSANP) via either an iv or ip injection. At one, 24, 48 and 96 h post-injection, four mice in each iv-/ip-injected group were sacrificed. The tissues/organs of interest (blood, heart, lung, liver, stomach, small intestine, large intestine, spleen, pancreas, kidney, muscle, urine, feces, bladder, bone, tumor, and ascites) were harvested and weighted, and the radioactivity of each tissue/organ was counted using a gamma counter. The accumulation of 111In-Au@HSANP in each tissue or organ was expressed as a percentage of the injected dose per gram (%ID/g).