Article pubs.acs.org/bc
Functionalized, Long-Circulating, and Ultrasmall Gold Nanocarriers for Overcoming the Barriers of Low Nanoparticle Delivery Efficiency and Poor Tumor Penetration Kate Y. J. Lee,‡ Gee Young Lee,‡ Lucas A. Lane,‡ Bin Li,† Jianquan Wang,† Qian Lu,† Yiqing Wang,*,†,‡ and Shuming Nie*,†,‡ †
Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu Province 210093, China ‡ Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, Atlanta, Georgia 30322, United States S Supporting Information *
ABSTRACT: The development of sophisticated nanoplatforms for in vivo targeted delivery of therapeutic agents to solid tumors has the potential for not only improving therapeutic efficacy but also minimizing systemic toxicity. However, the currently low delivery efficiency (about 1% of the injected dose) and the limited tumor penetration of nanoparticles remain two major challenges. Here we report a class of functionalized, long-circulating, and ultrasmall gold nanocarriers (5 nm gold core and 20 nm overall hydrodynamic diameter) for improved drug delivery and deep tumor penetration. By using doxorubicin as a model drug, our design also includes a pH-sensitive hydrazone linkage that is stable at neutral or slightly basic pH but is rapidly cleaved in the acidic tumor microenvironments and intracellular organelles. With a circulation halftime of 1.6 days, the small particle size is an important feature not only for efficient extravasation and accumulation via the enhanced permeability and retention (EPR) effect, but also for faster nanoparticle diffusion and improved tumor penetration. In xenograft animal models, the results demonstrate that up to 8% of the injected nanoparticles can be accumulated at the tumor sites, among the highest nanoparticle delivery efficiencies reported in the literature. Also, histopathological and direct visual examinations reveal dark-colored tumors with deep nanoparticle penetration and distribution throughout the tumor mass. In comparison with pure doxorubicin which is known to cause considerable heart, kidney, and lung toxicity, in vivo animal data indicate that this class of functionalized and ultrasmall gold nanoparticles indeed provides better therapeutic efficacies with no apparent toxicity in vital organs.
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One aspect having a large influence of a nanoparticle’s in vivo behavior is its size. Generally, for intravenously injected nanoparticles without any active tumor targeting ligands, larger sizes (50−100 nm) have longer circulation times and greater retention at the tumor site whereas smaller sizes exhibit higher perfusion throughout the tumor (5−10 nm).15,16 Thus, a compromise between these two size regimes (∼20 nm) may be optimal for the overall drugging of cancer cells throughout the tumor mass. However, typical polymeric or liposomal nanocarriers at this size range tend to have difficulty in fabrication
ne of the most important tasks in cancer nanomedicine is to design and develop nanosized drug carriers that can be used to favorably alter the pharmacokinetics and biodistribution of free drugs.1−5 The ultimate goals are to improve therapeutic efficacy and to reduce systemic toxicity and adverse effects. Extensive research in the last 20 years has led to many approaches and strategies such as the use of organic and inorganic nanoparticles as drug carriers,2,6 passive and active mechanisms for tumor targeting,7,8 multiple-stage “mothership” nanostructures,9,10 smart nanocluster bomblets,11 and stimuliresponsive nanoparticles for targeting the tumor habitats or microenvironments.12,13 However, the low nanoparticle delivery efficiency (less than 1% of the injected dose) and poor tumor penetrating are still two major problems.14 © 2016 American Chemical Society
Special Issue: Interfacing Inorganic Nanoparticles with Biology Received: May 5, 2016 Revised: June 23, 2016 Published: June 24, 2016 244
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Figure 1. Chemical synthesis and self-assembly of Au-DOX-PEG. (a) Chemical schematic of the synthesis of DOX-PDPH, and illustration showing the construction of the pH activateable nanocarrier using 5 nm gold cores. (b) Zeta potential of the nanocarriers. (c) Histogram of the particle sizes of the nanocarrier sample obtained by DLS. (d) TEM image of the gold core nanocarriers (scale bar = 10 nm).
sarcomas.18 Despite its high antitumor activity, doxorubicin exhibits systemic toxicity to normal tissues, notably inducing irreversible cardiotoxicity. In order to increase dosages to the tumor site and lower the systemic toxicity, nanocarriers of various formulations have been developed.19 Many of such formulations are effective in reducing systemic toxicities, but are limited in increasing the efficacy in treating the tumor compared to the free drug at equivalent dosage.20 Despite nanocarriers having greater tumor accumulation at the tumor site due to the enhanced permeation and retention effect arising from the leaky vasculature of the tumor microenvironment, typically the particles have sizes which prevent their perfusion throughout the tumor interstitium. As a result, the drugs may only be treating the cells at the tumor periphery leaving the cells more toward the center of the tumor mass unaffected. Smaller particle sizes will have greater perfusion; however, at sizes below 10 nm the circulation lifetimes can be significantly decreased as a result of renal clearance.21 Ideally the nanocarrier should have an intermediate size where it is large enough to have a long circulation time and a strong EPR effect, but also small enough that it can better perfuse the tumor mass. Recently, studies by Tang et al. have suggested that particle sizes near 20 nm are optimal in terms of total accumulation throughout the tumor.22 However, typical polymeric or liposomal nanocarriers can be difficult to synthesize in this size range and may be limited to low loading ration of the drug. To overcome these obstacles in creating 20nm-sized drug carriers with high payloads, we decided to
and/or have low drug payloads. Another aspect important to a nanocarrier’s performance is for the particle to release its payload only at the diseased site. This can be performed through a variety of chemical functionalities that release the drug through physiological cues (e.g., pH, enzymatic degradation, or redox state) from the tumor microenvironment.8,17 Here we present the utility of 5 nm gold particles as a facile option in generating ∼20 nm pH activated drug nanocarriers. The gold surface of the particles is easily functionalized with hydrazide linkers (3-[2-pyridyldithio]propionyl hydrazide, PDPH) which effectively release the anticancer drug doxorubicin (DOX) at low pH values and thiolated polyethene glycol (PEG) polymers to prevent biofouling and increase the overall particle size near 20 nm for optimal delivery to the tumor site. Additionally, the 5 nm size of the gold core is chosen due to being large enough to allow high surface availability for drug loading, but also small enough for eventual clearance from the body after completing its intended application. PEG-doxorubicin-PDPH-gold nanoparticle (Au-DOX-PEG) showed a prolonged blood circulation half time (1.6 days), and 8% of total injected gold was found in tumors.
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RESULTS AND DISCUSSION Chemical Synthesis and Characterization of Au-DOXPEG. Doxorubicin, an anthracycline derivative, is a commonly used chemotherapeutic agent for various malignancies such as solid tumors of the breast, esophagus, liver, and soft-tissue 245
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tends to “lie” close to the surface so that multiple points of a single polymer are covering the surface.28 Despite the low PEG surface density, the resulting Au-DOX-PEG was colloidally stable in various mediums such as salt and serum solutions (SI Figure S1). We suspect that in conjunction with DOX-PDPH coating, small-sized gold nanoparticle core (5 nm) and long chain length (MW = 5000) PEG resulted in a sufficient coverage of the gold surface for colloidal stability. It has been reported that nanoparticle stability increases with increasing PEG length and decreasing nanometer particle diameter.29 In particular, PEG with a molecular weight of ≥2000 Da can significantly reduce protein adsorption and result in colloidal stability in highly ionic strength media due to increased steric repulsive forces.30,31Supporting our results, Liu, Y. et al.29 found that (1) for any given PEG length, decrease in nanoparticle diameter resulted in a decreased amount of PEG per nanoparticle input to the reaction mixture and (2) for a given nanoparticle diameter, increase in PEG length resulted in a decrease in the amount of PEG amount input to the reaction mixture for colloidal stability. Furthermore, in accordance with DLVO theory, small-sized gold nanoparticles are more stable in general than the larger-sized gold nanoparticles due to minimized van der Waals attraction energy. Finally, we also wanted to ensure that bound doxorubicin is not affected by the addition of PEG. Similar to the method used for drug loading efficiency, the supernatant collected from centrifugation of various concentrations of PEG coated golddrug-PEG systems indicated that addition of PEG, especially an excess amount of PEG, did not affect bound doxorubicin and there was no detectable replacement of the bound drug. Drug Release Profile. Figure 2a shows the pH-dependent release profile of doxorubicin linked to the hydrazone bond of PDPH at 37 °C. Initial release of the drug from the
incorporate small 5 nm gold cores within our nanocarriers. We chose gold nanoparticles due to their desirable properties of being bioinert, having high monodispersity, and having a large surface which is easily functionalized with a variety of chemical moieties by simple thiol anchoring. Additionally, it is important that the nanocarrier only release its payload once it has reached the diseased tissue site. As an added measure to improve the therapeutic efficacies of our nanocarriers, we have incorporated pH sensitive PDPH hydrazide linkers to DOX to prevent premature release of the drug. The hydrazone bond is stable under neutral pH conditions, but it is cleaved under mild acidic conditions,23 resembling the acidic tumor microenvironement, the endosomal and lysosomal environment. In addition to providing pH sensitivity, the PDPH linker provides thiol anchoring of the drug complex to the surface of the gold nanoparticle. Our pH activatable nanocarrier was synthesized by first coating 5 nm gold nanoparticles with DOX-PDPH, then using the remainder of the particle surface for grating thiolated PEG molecules (Figure 1a). The PEG polymers increase the circulation time of nanoparticles by reducing opsinization of the nanoparticles.24 Furthermore, the PEG coating increases the hydrodynamic size of the 5 nm gold nanoparticles to ∼17.8 ± 1.3 nm which is near the optimal size range for delivery of nanoparticles to tumors. To find the maximum drug loading capacity of 5 nm gold nanoparticle while maintaining the colloidal stability of gold nanoparticle, UV−vis spectroscopy and fluorescence spectra were used to test the adsorption of series of different concentrations of DOX-PDPH onto gold. When DOX-PDPH was conjugated to gold nanoparticles in water, the fluorescence of doxorubicin was quenched on gold surface (SI Figure S2 “Before Centrifuge”). Previous studies also report quenching of fluorescent dyes on metallic particles when they are chemisorbed onto the surface. Furthermore, fluorescence quenching on the metallic surface is observed for the distance of a few nanometers,25−27 which suggests the proximity of doxorubicin onto the gold surface linked via short PDPH linker. When the gold nanoparticle−anticancer agent−PEG system was centrifuged for purification, the supernatant did not contain any detectable amount of unbound doxorubicin up to 5.5 wt %. However, when excess DOX-PDPH was added to the gold nanoparticle solution, we observed fluorescence of unbound DOX-PDPDH in the supernatant (SI Figure S2(c)). Thus, the maximum drug loading capacity of 5 nm gold nanoparticles was ∼5.5 wt %. Although it looks not very impressive in terms of drug loading in weight percent, considering the huge density difference between gold (19.32 g/cm3) and polymers (usually less than 1.0 g/cm3), the number of drug molecules per particle of Au-DOX-PEG is much greater than polymeric nanoparticle counterpartners. To find the optimum PEG density for colloidal gold stability, dynamic light scattering measurement was used for size change. After subtracting the gold surface area occupied by modified doxorubicin, the available surface area was coated with various concentrations of PEG. As we increased the PEG concentration, the saturation point was reached for gold-drug-PEG size measurement (Table S1). Saturation of the gold nanoparticle surface at low % PEG values indicate that most of the gold surface is coated with modified doxorubicin, and PEG is bound onto the gold surface in a “mushroom conformation”. Mushroom conformation is characterized by low surface grafting density and the polymer
Figure 2. (a) pH-Sensitive drug release of Au-DOX-PEG in neutral and acidic conditions. (b) In vitro therapeutic efficacy of Au-DOXPEG:MTT assay results with 4T1 murine breast cancer cells (equal amount of gold was used for Au-DOX-PEG and Au-PEG groups; 7 μg/mL of doxorubicin and equivalent was used for doxorubicin and Au-DOX-PEG, respectively). 246
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Bioconjugate Chemistry nanoparticles ( 0.05; #, p < 0. 01; §, p > 0.05; pink arrow indicates the time points of treatment); tumor weight (**: p < 0.05 from control only). (c) Extracted tumor raw weight. n = 5 and standard error of mean were used for all figures.
Figure 4. In vivo results of Au-DOX-PEG within tumor: Collection of extracted tumor from control, doxorubicin, Au-DOX-PEG, and AuPEG groups. Bottom picture indicates the halved Au-DOX-PEG tumor.
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increases effectiveness of the nanocarrier to treat cells deeper within the tumor. We observed a significant accumulation of Au-DOX-PEG in the tumor compared to other normal organs (Figure 6). For our Au-DOX-PEG system, up to 6.8% of the
Figure 6. ICP-MS analysis results: high accumulation of Au-DOXPEG at tumor site compared to other nontumor sites.
total injected gold (4 injections) was taken up by the tumor. If we are only concerned with Au NP accumulation in the tumor, Au-PEG (no doxorubicin) data can be further leveraged to have more data points. Statistically, there were no significant differences among the gold amount in the tumor between Au-PEG and Au-DOX-PEG (see Table S2 in SI for detailed calculation). In combined data obtained from both Au-DOXPEG and Au-PEG, the average total tumor accumulation of Au NPs reaches 8.3%. Different from most of other reported cases, our data were collected 7 days after fourth injections; the results may further increase if we sacrifice the mice earlier. The high accumulation and uptake of Au-DOX-PEG is rendered from the size of our system (5 nm core and ∼20 nm for coated). However, we cannot exclude the fact that the higher doses used in this study may have altered the accumulation rate of Au-DOX-PEG at the tumor site, which could have led to increased nonspecific uptake of our system by the cancer cells. Au-DOX-PEG nanoparticles also accumulated in spleen, liver, kidney, heart, and lung (Figure 7); however, no significant toxicity was observed. Evaluation of Systemic Toxicity. In addition to the increased therapeutic efficacies of nanocarriers, they also have the ability to reduce the systemic toxicity of the free drug. Compared to pure doxorubicin, the Au-DOX-PEG system lacked any apparent toxicity in any of the vital organs including the spleen, liver, heart, kidney, and lung. Free DOX is known for its cardiotoxicity, but it is however less toxic to the RES organs of the liver and spleen. Thus, nanocarriers which generally have increased uptake by the RES compared to the free drug will reduce the overall systemic toxicity of DOX. The inflammatory and oxidative response can be obtained by analyzing the blood serum. For example, when there is damage to the liver, there is an increased expression of certain immunological proteins in the blood that can be detected by serum analysis. Serum protein analysis results seen in Figure 8 complement our toxicity results found in histological results (Figure S3). Serum proteins such as ALT, AST, and Alkaline phosphatase can be used to measure liver toxicity. Here, there were no significant differences among control (untreated), doxorubicin, Au-DOX-PEG, and Au-PEG groups for each ALT,
Figure 5. Spatial distribution of Au-DOX-PEG within tumor: (a) brightfield and darkfield microscopy images of Au-DOX-PEG present within tumor (arrows and orange chunks indicates Au-DOX-PEG); (b) TEM images of Au-DOX-PEG inside the tumor cell; (c) TEM images of Au-DOX-PEG outside the tumor cell.
particles are found inside vacuoles or endosome-looking vesicles within the cells. Pharmacokinetics and Biodistribution of Au-DOXPEG. Pharmacokinetic and biodistribution analysis of Au-DOXPEG in the tumor and various organs was performed using inductive coupled plasma mass spectroscopy (ICP-MS). The Au-DOX-PEG had a prolonged blood circulation half-time of ∼1.6 days compared to that of pure doxorubicin being a few minutes. The prolonged circulation time is attributed to the size of the nanoparticle system preventing widespread systemic release of the drug within the body and hindering clearance through renal excretion. Additionally, the prolonged circulation can be attributed to the PEG coating which hinders opsonization and subsequent clearance by the RES organs. Such prolonged increases in circulation are highly desired in therapeutic applications as the increased concentrations of the therapeutic agents in the blood creates a stronger driving force for particle permeation into the tumor interstitium. This in turn 248
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Figure 7. TEM images of various organs after Au-DOX-PEG administration. Spleen: Red blood cells are seen (polychromatophilic). Liver: Dark spots are glycogen granules. Gold nanoparticles are mostly inside the blood vessel cells (outside the cell) and some are seen inside the liver cell near the nucleus. Kidney: Some are inside the blood vessel cell and others are inside some cells. Heart: Gold nanoparticles are inside an irregular-shaped cell in between the heart muscle fibers. Some gold nanoparticles are in the cavity/muscle lignin of the heart. Lung: Gold nanoparticles are inside the blood vessel (outside the lung cell).
toxicities seen in the doxorubicin group are further complemented with the serum protein profile results. Kidney toxicity can be measured through total bilirubicin, creatinine,
AST, and alkaline phosphatase level. This indicates minimal toxicity to the liver, which is consistent with the histology results (shown in Figure S3). Similarly, kidney and heart 249
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from Pierce (Rockford, IL). Poly(ethylene glycol) (CH3OPEG-SH) of molecular weight 5000 was purchased from Rapp Polymere (Germany). Methanol, acetonitrile, dimethyl sulfoxide, citric acid, and MTT based in vitro toxicology assay kit were all obtained from Sigma (St. Louis, MO). Milli-Q deionized water (Millipore, 18.2 MΩ cm−1) was used throughout the experiments. All of the products were used without modification or purification unless otherwise noted. Instrumentation. Nanoparticle surface charge (zeta potential) and size were measured by the ZetaSizer NanoZS90 (Malvern Instrument). Adsorption spectra were obtained through UV−vis spectrophotometer (Beckman Coulter DU530). Fluorescence of nanoparticles were evaluated by a Fluoromax-2 (Jobin Yvon-Spex, Horiba Group) fluorometer, equipped with a xenon arc lamp. A scanning multiwell spectrometer, Synergy 2 (Biotek), was used to read the absorption of blue formazan crystals for MTT assays. Gold content was analyzed by ICP-MS (HP 4500, Agilent Technologies). TEM were taken by using Hitachi H7500 high-magnification electron microscope. Finally, an Olympus IX71 inverted microscope was used to take brightfield and darkfield images. Cell Line and Mouse Model. The murine breast cancer cell line 4T1 was obtained as a gift from Dr. Lily Yang (Emory University). The 4T1 cells were cultured in RPMI-1640 (Mediatech, Inc.; Manassas, VA) containing 10% fetal bovine serum (American Type Culture Collection; Manassas, VA) and penicillin−streptomycin solution (Mediatech, Inc.; Manassas, VA). Cells were grown in a 37 °C humidified incubator containing 5% CO2. 1× phosphate buffered saline (1× PBS) was purchased from Mediatech, Inc. 6−7 week old female Balb/C mice were obtained from a commercial vendor (Jackson Laboratories). The protocols used within this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University. Statistical Analysis. Statistical analysis was performed using one-way ANOVA followed by a multiple comparison Bonferroni’s test. Data were collected from at least three different animals and P value
