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Quantitative Examination of the Active Targeting Effect# the Key Factor for Maximal Tumor Accumulation and Retention of Short-Circulated Biopolymeric-Nanocarriers Jianquan Wang, Gee Young Lee, Qian Lu, Xianghong Peng, Jiangxiao Wu, Siyuan Wu, Brad A. Kairdolf, Shuming Nie, Yiqing Wang, and Lucas A. Lane Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017
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Bioconjugate Chemistry
Quantitative Examination of the Active Targeting Effect： ：the Key Factor for Maximal Tumor Accumulation and Retention of ShortCirculated Biopolymeric-Nanocarriers Jianquan Wang, †, § Gee Young Lee, ‡, § Qian Lu, † Xianghong Peng, ‡ Jiangxiao Wu, † Siyuan Wu, † Brad A. Kairdolf, ‡ Shuming Nie, †,‡ Yiqing Wang*,†,‡ and Lucas A. Lane *,† †
Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu Province 210093, China. ‡ Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA 30322, USA. ABSTRACT: Targeted and non-targeted biopolymeric nanoparticles with identical hydrodynamic sizes and surface charges were quantitatively examined in terms of the pharmacokinetic and biodistribution differences in detail. In adding cancer cell targeting folate molecules to the surface of the heparin nanocarriers, the amount of drug delivered to the tumor is doubled and tumor growth inhibition is significantly enhanced. The folate-targeted heparin particles offered similar therapeutic potentials compared to their synthetic long-circulating analogues, thus presenting a viable alternative for drug delivery vehicle construction using biological polymers which are easier for the body to eliminate.
INTRODUCTION A majority of clinically approved anticancer drugs are small molecular compounds. Once intravenously injected into the patient’s bloodstream, they tend to have wide biodistributions among various tissues that are not intended targets and have rapid renal elimination. This results in widespread systemic toxicity, low bioavailability, and suboptimal efficacy of these compounds. The development of nanoparticle drug carriers aims to overcome the limitations of the administration of free small molecule drugs and have shown great promise in increasing bioavailability and drug payload to tumor target sites.1-3 The strategy in the delivery of nanoparticles to tumors typically falls into one of two categories: passive or active targeting. For passive targeting, nanoparticles with size ranges between ten to a few hundred nanometers tend to accumulate within tumors due to the enhanced permeation and retention (EPR) effect which is a result of the tumor’s leaky vasculature and impaired lymphatics.4 For active targeting, in addition to the EPR effect the nanoparticle surfaces are chemically modified with moieties that bind specifically to tumor-associated membrane components and aid in the intracellular uptake of the particles.5,6 Conventional wisdom in the design of nanocarriers has been that in order to increase accumulation within the tumor site, long blood circulation times of the intravenously injected agents are necessary. Achieving long circulation times typically involves the use of synthetic hydrophilic polymers on the surface such as polyethylene glycol. However, a number of reports have shown that the use of active targeting on particles having long circulation times, such as those with polyethylene glycol (PEG) coatings, offers little benefits over their passive counterparts.5,6
Less attention has been paid to the extent of active targeting on nanoparticles with shorter circulation times. Nanoparticles composed of naturally occurring biopolymers, such as chitosan, gelatin, and heparin, are more desirable for drug delivery because those biopolymers are synthesized by living organisms, which can have greater biocompatibility as they are more easily degraded than their synthetic counterparts but tend to be neglected as drug delivery vehicles as a result of their short circulation times. However, it should be noted that a short circulation time does not equate with ineffective therapeutic efficacies for all cases of nano-sized drug carriers.7,8 In this study we demonstrate the significant increases in therapeutic efficacy that active targeting has over passive in biopolymeric nanocarriers, which is typically not seen in their synthetic counterparts. Two types of nanoparticles composed of heparin biopolymers delivering the anticancer drug cisplatin (cis-diamminedichloroplatinum, DDP) are compared: a passive targeting nanoparticle consisting of heparin-DDP conjugates (HDDP), and an active targeting nanoparticle which includes folate molecules on the surface (HFDDP). Using in vivo models, we precisely determine the properties of blood circulation time, biodistribution, and drug accumulation in the tumor mass of both targeting types. We found that by adding cancer cell targeting folate molecules to the surface of heparin nanocarriers, the amount of drug delivered to the tumors is doubled and tumor growth inhibition is significantly enhanced while systemic toxicity is further decreased. RESULTS AND DISCUSSION DDP is a widely used anticancer drug for a variety of cancers. Despite its prevalent use within the clinic, the free drug suffers from a limited ability to localize to tumoral tissues from the blood and causes severe systemic toxicity.9,10 To increase the
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tumor localization of the drug and decrease off target toxicity, nanocarriers of several varieties have been employed. Among the several nanocarrier classes, most rely on synthetic polymers for their construction, such as PEG, to increase circulation times for maximal tumor accumulation.11 However, less focus has been paid to biopolymers for nanocarrier construction as they typically have much shorter circulation times and therefore are deemed to have lower tumor accumulation. Recently, our group has shown that nanocarriers made of the biopolymers, though having short circulation times, still offer high therapeutic efficacies and much lower systemic toxicities than the free drug. 7,8 Specifically, nanocarriers composed of heparin biopolymers can offer advantages over synthetic analogues due the polymer having FDA approval for the treatments of various diseases and its ease of biodegradation.12
Figure 1. Preparation of Heparin-DDP (HDDP) and HeparinFolate-DDP (HFDDP) As shown in Figure 1., Heparin and DDP molecules are able to self-assemble into nanoparticles by the coordination between the heparin carboxyl groups and the central Pt ion of DDP via substitution of two chlorides. Mixing molar ratios of 1:1 unfractionated heparin to DDP under gentle stirring in water led to narrow dispersed nanoparticles with an average size 150 ± 25 nm after 24 hours. To further increase the DDP loading capacity and reduce the undesirable anticoagulant property of heparin, we modified hydroxyl groups to carboxyls with succinic anhydride according to a previously reported procedure leaving the polymers with 20 wt% of succinyl groups.12 The succinyl groups also allowed further conjugation of folate targeting molecules to the polymers through amide linkages. Adding folate to the modified heparin polymers at a ratio of 1:10 led to a 5 wt% of folate content.13 Both HDDP and HFDDP contained about 23 wt% DDP, which was measured by ICP-MS. HDDP and HFDDP showed similar hydrodynamic size (20 ± 5 nm) and surface charge (~5 mV). Table 1. Cellular uptake of free DDP, HDDP and HFDDP at different concentrations (0.05-0.4 µg/mL, DDP equivalence) after 2 h (KB-3-1 cells). dose
DDP(ng)
HDDP(ng)
HFDDP(ng)
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0.05 µg/mL
0.81±0.17
1.31±0.12
1.87±0.45
0.1 µg/mL
0.89±0.25
1.81±0.31
2.9±0.34
0.2 µg/mL
1.35±0.42
2.2±0.24
7.2±1.2
0.4 µg/mL
1.8±0.54
6.17±1.26
16.97±1.87
Free DDP is known to have slow diffusion through the cellular membrane which limits its therapeutic potential at its target.14 Nanoparticles can increase payloads of DDP by moving through the more rapid internalization processes of receptoror non-receptor- mediated endocytosis. Receptor-mediated endocytosis, where targeting ligands bind to specific receptors on the cell membrane and trigger an uptake response, tends to have faster internalization kinetics than non-receptor-mediated processes for similar particle constructs.15 To test the dependence of the amount of DDP delivered intracellularly on the route of internalization, equivalent drug dosings were introduced to cell cultures using free DDP and using the nontargeted and targeted HDDP and HFDDP nanocarriers, respectively. Here two cell lines were tested: KB-3-1, which overexpresses the folate receptor, and TU212, which has negative expression for the receptor. The intracellular drug concentrations were determined by the amount of Pt by ICP-MS analysis. To minimize potential interference from dead cells and extracellular release of DDP from the nanoparticles, incubation times were limited to 2 hours, where 90% of the DDP is still within the nanocarriers. At a dose of 0.4 µg/mL, DDP delivered by the actively targeted nanoparticles ([PtHFDDP]) had 9 times the intracellular concentration of the drug compared to the free drug ([PtDDP]) and near 3 times that of the nontargeted ([PtHDDP]) nanoparticle administration routes with detected Pt levels (See Table 1).
Figure 2. SRB assay in KB-3-1 cells treated with free DDP, HDDP and HFDDP at a concentration of 0.5 µg/mL (DDP equivalence). *P
