- Nano Express
- Open Access
A Comparative Evaluation of Hydroxycamptothecin Drug Nanorods With and Without Methotrexate Prodrug Functionalization for Drug Delivery
- Fuqiang Guo†1,
- Zhongxiong Fan†1,
- Jinbin Yang3,
- Yang Li2,
- Yange Wang2,
- Hai Zhao1,
- Liya Xie4Email author and
- Zhenqing Hou1, 2Email authorView ORCID ID profile
© The Author(s). 2016
- Received: 10 May 2016
- Accepted: 19 August 2016
- Published: 31 August 2016
We developed a novel self-targeted multi-drug co-delivery system based on rod-shaped 10-hydroxycamptothecin (CPT) nanoanticancer drug (CPT NRs) followed by a surface functionalization with self-targeting PEGylated lipid-conjugated methotrexate (MTX) pro-anticancer drug. The self-targeting effect and in vitro cell viability of the MTX-PEG-CPT NRs on HeLa cells were demonstrated by comparative cellular uptake and MTT assay of the PEG-CPT NRs. In vitro studies showed the feasibility of using this high drug-loading MTX-PEG-CPT NRs in self-targeted drug delivery, controlled-/sustained-release, and synergistic cancer therapy. More importantly, this work would stimulate interest in the use of PEGylated lipid-conjugated MTX by introducing an early-phase tumor-targeting role and then driving a late-phase anticancer role for the highly convergent design of nanomulti-drug, which may advantageously offer a new and simple strategy for simultaneously targeting and treating FA receptor-overexpressing cancer cells.
- Self-targeted multi-drug co-delivery system
- Controlled and sustained release
Combination therapy has been considered to be a promising strategy for cancer treatment [1, 2]. Nanoparticles with multi-therapeutic actions targeted to different action sites of target cell could more effectively reduce the blood/renal clearance, increase the tumor accumulation, and, more importantly, induce the apoptosis/death and inhibit the growth of cancer cells . In recent years, nanoparticles with chemo-chemo, chemo-gene, chemo-thermal, and chemo-photodynamic combination therapies have attracted considerable attention . Especially, thousands of nanoparticles with chem-chem combination therapy have been widely used in treatment of many types of tumor. For the combination cancer chemotherapy, to achieve the synergistic anticancer effect, overcome the drug resistance, and reduce the side effects, multi-anticancer drugs have been co-loaded within the nanoscaled drug carriers of inert carrier materials and co-delivered to the tumor. However, when these drug carriers with comparatively low drug-loading ability (typically < 20 %) were clinically used for cancer treatment, the considerable decrease of effective accumulation of drug would occurred at the tumor site; in addition, large amounts of inert drug carriers could impose additional burden on the patients such as dosage-related systemic toxicity, metabolism/excretion/degradation concern, and serious inflammation [5, 6].
Anticancer drug , anticancer drug-anticancer drug physical complex , and anticancer drug-anticancer drug chemical conjugate [9, 10] as a material to construct the drug nanocrystals or nanoparticles (carrier-free nanoscaled drug system, self-delivery nanodrug) without (or with little) addition of any inert carrier materials have become attractive and have demonstrated the superior performance in the field of drug delivery for cancer treatment. However, insufficient bioenvironmental stability  or poor-targeting efficiency seriously limited its further clinical application. Our previous study demonstrated that methotrexate (MTX) anticancer drug itself could also be utilized as a folic acid (FA) receptor-overexpressing cancer cell-specific “targeting ligand,” playing a dual role [12, 13]. Inspired and motivated by the unique advantage of drug self-constructed nanoparticles and potential merits of therapeutic agent with a dual role, one would envisage the possibility of designing a self-targeting and high-drug loading nanomulti-drug to simply achieve the more effectively and specifically cell-targeting effect and cell-killing efficacy against cancer cell. The highly convergent design of self-targeting nanomulti-drug by “self-targeted multi-drug co-delivery and combination cancer therapy” may open a promising new door to nanomedicine.
To obtain CPT-MTX composite nanomedicine, CPT was firstly dissolved in ethanol, and then, the mixture was added to water under ultrasonication for solvent exchange; finally, the resulted rod-shaped CPT drug nanoparticles (CPT NRs) were absorbed by DSPE-PEG-MTX (its structure was confirmed by 1H nuclear magnetic resonance (1H NMR, Additional file 1: Figure S1, ESI†), Fourier transform infrared spectroscopy (FTIR, Additional file 1: Figure S2, ESI†), and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, Additional file 1: Figure S3, ESI†) via hydrophobic interactions between the hydrophobic surface of NRs and the long-chain fatty acids of phospholipid of DSPE-PEG-MTX (designed as MTX-PEG-CPT NRs)). During the preparing process, ultrasonication was used to accelerate the diffusion of ethanol into water, resulting in the strong supersaturation and high nucleation rates to produce uniform nanosuspension. The addition of self-targeting PEGylated lipid prodrug to the hydrophilic region of the CPT NRs by non-covalent association resulted in the production of CPT NRs core-MTX shell-constructing MTX-PEG-CPT NRs with good water dispersibility, stability, and long-circulating ability.
To evaluate the bioenvironmental stability of the MTX-PEG-CPT NRs, we determined the average hydrodynamic particle size and fluorescence intensity of the MTX-PEG-CPT NRs in phosphate-buffered saline (PBS) during 3 days of incubation period by DLS and fluorescence analysis (Fig. 3e, f). PEG-CPT NRs and MTX-PEG-CPT NRs show a no significant increase of average hydrodynamic particle size and decrease of fluorescence intensity. These results indicated the PEG-CPT NRs and MTX-PEG-CPT NRs possessed good bioenvironmental stability, whereas the CPT NRs observably form the large agglomerates and aggregation because of the charge elimination on the surface of CPT NRs by zwitterionic phosphonates of PBS. In addition, the MTX-PEG-CPT NRs also showed no significant change of hydrodynamic particle size and fluorescence intensity in Dulbecco’s modified Eagle medium with fetal bovine serum (10 %) (Additional file 1: Figure S4, ESI†). These results demonstrated the protecting effect of PEG on the MTX-PEG-CPT NRs against the zwitterion effect and protein adsorption by the combination of hydration effect and steric effect [23, 24].
The drug release profile of the MTX-PEG-CPT NRs is of great importance for multi-drug co-delivery applications. As shown in Fig. 3g, during the first phase of 8 h, the accumulative CPT release of the MTX-PEG-CPT NRs exhibited about 25 %. And both of these CPT NRs exhibited a similar CPT release behavior, which indicated the introduction of MTX-targeting ligand/anticancer drug did not alter the drug release of PEGylated CPT NRs with long-chain-length PEG. It was worthy noted that these two types of CPT NRs exhibited no early-phase serious burst release but a sustained and steady release characteristic. The result was explained by the fact that CPT with the crystalline state but not the amorphous one effectively avoids an unstable spatial arrangement, which is essential for their in vivo applications because of the reduced drug burst release in blood circulation, enhanced blood circulation effect, and improved tumor-targeted drug delivery.
We further studied the MTX release of the MTX-PEG-CPT NRs in PBS with different pH values in the presence or absence of proteases (pH 7.4 without proteases simulates blood circulation conditions, pH 5.0 with proteases simulates tumor cells lysosome). As depicted in Fig. 3h, at pH 7.4 in the absence of proteases, the accumulative MTX release of the MTX-PEG-CPT NRs at 24 h reached no more than 10 %. In sharp contrast, the addition of enzyme as well as the decrease of pH significantly promoted the MTX release from the MTX-PEG-CPT NRs. For instance, at pH 5.0 in the presence of proteases, the accumulative release of MTX greatly increased to about 45 % 24 h. This result indicated that the chemical linkage between MTX and MTX-PEG-CPT NRs was cleavaged by the combination of enzyme and pH to release the active MTX drug, “turn off” the targeting role of MTX while “turn on” the anticancer role of MTX. These also showed the potential for the in vivo applications such as the more effective multi-drug co-delivery to tumor microenvironment and tumor cells with reduced drug leakage/burst release in blood circulation.
We investigated the cellular uptake of the MTX-PEG-CPT NRs by HeLa cells using laser confocal scanning microscopy (CLSM). The qualitative result indicated that the functionalization of MTX improve the cellular internalization of the MTX-PEG-CPT NRs in comparison with the PEG-CPT NRs (Fig. 4). We further evaluated the cellular uptake of the MTX-PEG-CPT NRs by HeLa cells by flow cytometry. The quantitative results indicated the significantly higher cellular uptake efficacy of the MTX-PEG-CPT NRs compared with the PEG-CPT NRs (Fig. 4b). All results proved that self-targeting MTX moiety enhanced the target efficacy to potentially help the MTX-PEG-CPT NRs enter FA receptor-overexpressing HeLa cells by FA receptor-mediated endocytosis via multivalent receptor-ligand interactions .
In summary, the self-targeting, controlled-/sustained-release, and multi-drug-loaded MTX-PEG-CPT NRs have been prepared for highly effective “self-targeted multi-drug co-delivery and combination cancer therapy.” The MTX-PEG-CPT NRs can be specifically uptaken by cancer cells, which result in an efficient intracellular both drug concentration and excellent cytotoxicity. More importantly, the MTX-PEG-CPT NRs can kill cancer cells through different functional roles, action sites, and anticancer mechanisms of both MTX and CPT, achieving a synergy in anticancer activity and showing a potential for clinical treatment of nanomedicine.
Fuqiang Guo acknowledges the financial support by the Natural Science Foundation of China (Grant No. 21502007). The project was supported by the Natural Science Foundation of Fujian Province of China (No.2016J01406), Fujian Province medical innovation project (2014-CX-35), and science and technology personnel training project Xinjiang Uygur Autonomous Region of China (qn2015bs014).
FG and ZF conceived and carried out the experiments, analyzed the data, and wrote the paper. ZH designed the study, supervised the project, analyzed the data, and wrote the paper. JY, YL, and YW assisted in the synthesis and characterizations of the NPs. HZ and LX assisted in the biological evaluations of the NPs. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Woodcock J, Griffin JP, Behrman RE (2011) Development of novel combination therapies. N Engl J Med 364:985–987View ArticleGoogle Scholar
- Lehar J, Krueger AS, Avery W, Heilbut AM, Johansen LM, Price ER, Rickles RJ, Short GF 3rd, Staunton JE, Jin X, Lee MS, Zimmermann GR, Borisy AA (2009) Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat Biotechnol 27:659–666View ArticleGoogle Scholar
- Liu Y, Fang J, Kim YJ, Wong MK, Wang P (2014) Codelivery of doxorubicin and paclitaxel by cross-linked multilamellar liposome enables synergistic antitumor activity. Mol Pharmaceutics 11:1651–1661View ArticleGoogle Scholar
- Deng X, Liang Y, Peng X, Su T, Luo S, Cao J, Gu Z, He B (2015) A facile strategy to generate polymeric nanoparticles for synergistic chemo-photodynamic therapy. Chem Commun 51:4271–4274View ArticleGoogle Scholar
- Shen Y, Jin E, Zhang B, Murphy CJ, Sui M, Zhao J, Wang J, Tang J, Fan M, Van Kirk E, Murdoch WJ (2010) Prodrugs forming high drug loading multifunctional nanocapsules for intracellular cancer drug delivery. J Am Chem Soc 132:4259–4265View ArticleGoogle Scholar
- Yu C, Zhou M, Zhang X, Wei W, Chen X, Zhang X (2015) Smart doxorubicin nanoparticles with high drug payload for enhanced chemotherapy against drug resistance and cancer diagnosis. Nanoscale 7:5683–5690View ArticleGoogle Scholar
- Fuhrmann K, Gauthier MA, Leroux JC (2014) Targeting of injectable drug nanocrystals. Mol Pharmaceutics 11:1762–1771View ArticleGoogle Scholar
- Chen F, Zhao Y, Pan Y, Xue X, Zhang X, Kumar A, Liang XJ (2015) Synergistically enhanced therapeutic effect of a carrier-free HCPT/Dox nanodrug on breast cancer cells through improved cellular drug accumulation. Mol Pharmaceutics 12(7):2237-2244.Google Scholar
- Zhang T, Huang P, Shi L, Su Y, Zhou L, Zhu X, Yan D (2015) Self-assembled nanoparticles of amphiphilic twin drug from floxuridine and bendamustine for cancer therapy. Mol Pharmaceutics 12(7):2328-2336.Google Scholar
- Huang P, Hu M, Zhou L, Wang Y, Pang Y, Tong G, Huang W, Su Y, Zhu X (2015) Self-delivery nanoparticles from an amphiphilic covalent drug couple of irinotecan and bendamustine for cancer combination chemotherapy. RSC Adv 5:86254–86264View ArticleGoogle Scholar
- Li Y, Wu Z, He W, Qin C, Yao J, Zhou J, Yin L (2015) Globular protein-coated paclitaxel nanosuspensions: interaction mechanism, direct cytosolic delivery, and significant improvement in pharmacokinetics. Mol Pharmaceutics 12:1485–1500View ArticleGoogle Scholar
- Li Y, Lin J, Wu H, Jia M, Yuan C, Chang Y, Hou Z, Dai L (2014) Novel methotrexate prodrug-targeted drug delivery system based on PEG-lipid-PLA hybrid nanoparticles for enhanced anticancer efficacy and reduced toxicity of mitomycin C. J Mater Chem B 2:6534–6548View ArticleGoogle Scholar
- Li Y, Lin J, Wu H, Chang Y, Yuan C, Liu C, Wang S, Hou Z, Dai L (2015) Orthogonally functionalized nanoscale micelles for active targeted codelivery of methotrexate and mitomycin C with synergistic anticancer effect. Mol Pharmaceutics 12:769–782View ArticleGoogle Scholar
- Jain S, Jain R, Das M, Agrawal AK, Thanki K, Kushwah V (2014) Combinatorial bio-conjugation of gemcitabine and curcumin enables dual drug delivery with synergistic anticancer efficacy and reduced toxicity. RSC Adv 4:29193–29201View ArticleGoogle Scholar
- Roberts GC, Feeney J, Birdsall B, Charlton P, Young D (1980) Methotrexate binding to dihydrofolate reductase. Nature 286:309View ArticleGoogle Scholar
- Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA (1966) Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from camptotheca acuminata1,2. J Am Chem Soc 88:3888–3890View ArticleGoogle Scholar
- Li Y, Lin J, Huang Y, Li Y, Yang X, Wu H, Wu S, Xie L, Dai L, Hou Z (2015) Self-targeted, shape-assisted, and controlled-release self-delivery nanodrug for synergistic targeting/anticancer effect of cytoplasm and nucleus of cancer cells. ACS Appl Mater Interfaces 7:25553–25559View ArticleGoogle Scholar
- Chauhan VP, Popovic Z, Chen O, Cui J, Fukumura D, Bawendi MG, Jain RK (2011) Fluorescent nanorods and nanospheres for real-time in vivo probing of nanoparticle shape-dependent tumor penetration. Angew Chem Int Ed 50:11417–11420View ArticleGoogle Scholar
- Gratton SE, Ropp PA, Pohlhaus PD, Luft JC, Madden VJ, Napier ME, DeSimone JM (2008) The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci U S A 105:11613–11618View ArticleGoogle Scholar
- Cui F, Lin J, Li Y, Li Y, Wu H, Yu F, Jia M, Yang X, Wu S, Xie L, Ye S, Luo F, Hou Z (2015) Bacillus-shape design of polymer based drug delivery systems with Janus-faced function for synergistic targeted drug delivery and more effective cancer therapy. Mol Pharmaceutics 12:1318–1327View ArticleGoogle Scholar
- Cho K, Wang X, Nie S, Chen ZG, Shin DM (2008) Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Res 14:1310–1316View ArticleGoogle Scholar
- Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA (2008) Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A 105:14265–14270View ArticleGoogle Scholar
- Li Y, Wu H, Yang X, Jia M, Li Y, Huang Y, Lin J, Wu S, Hou Z (2014) Mitomycin C-soybean phosphatidylcholine complex-loaded self-assembled PEG-lipid-PLA hybrid nanoparticles for targeted drug delivery and dual-controlled drug release. Mol Pharmaceutics 11:2915–2927View ArticleGoogle Scholar
- Li Y, Lin J, Yang X, Li Y, Wu S, Huang Y, Ye S, Xie L, Dai L, Hou Z (2015) Self-assembled nanoparticles based on amphiphilic anticancer drug-phospholipid complex for targeted drug delivery and intracellular dual-controlled release. ACS Appl Mater Interfaces 7:17573–17581View ArticleGoogle Scholar
- Lin J, Li Y, Li Y, Wu H, Yu F, Zhou S, Xie L, Luo F, Lin C, Hou Z (2015) Drug/dye-loaded, multifunctional PEG-chitosan-iron oxide nanocomposites for methotraxate synergistically self-targeted cancer therapy and dual model imaging. ACS Appl Mater Interfaces 7:11908–11920View ArticleGoogle Scholar
- Lin J, Li Y, Li Y, Cui F, Yu F, Wu H, Xie L, Luo F, Hou Z, Lin C (2015) Self-targeted, bacillus-shaped, and controlled-release methotrexate prodrug polymeric nanoparticles for intratumoral administration with improved therapeutic efficacy in tumor-bearing mice. J Mater Chem B 3:7707-7717Google Scholar
- Martinez-Veracoechea FJ, Frenkel D (2011) Designing super selectivity in multivalent nano-particle binding. Proc Natl Acad Sci U S A 108:10963–10968View ArticleGoogle Scholar
- Chou T-C (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70:440–446View ArticleGoogle Scholar