Nanoparticles are usually prepared from natural or synthetic polymeric materials using supercritical fluid technology [16York P, Kompella UB, Shekunov BY. Supercritical fluid technology for drug product development. New York: Marcel Dekker 2004.
[http://dx.doi.org/10.1201/9780203021378] ], the solvent evaporation technique [17Lee M, Cho YW, Park JH, et al. Size control of self-assembled nanoparticles by an emulsion/solvent evaporation method. Colloid Polym Sci 2006; 284(5): 506-12.
[http://dx.doi.org/10.1007/s00396-005-1413-3] ], and high pressure homogenization [18Wan X, Zheng X, Pang X, et al. The potential use of lapatinib-loaded human serum albumin nanoparticles in the treatment of triple-negative breast cancer. Int J Pharm 2015; 484(1-2): 16-28.
[http://dx.doi.org/10.1016/j.ijpharm.2015.02.037] [PMID: 25700543] ]. Ideal polymeric materials should be biodegradable and biocompatible. Examples of natural polymers include gelatin, chitosan, alginate, gliadin, etc., while those of synthetic polymers include polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic) acid (PLGA), poly (alkylcyanoacrylate) (PACA), etc. However, some non-biodegradable polymeric materials can also be used for the preparation of nanoparticles such as polymethyl methacrylate (PMA) and polymethyl methacrylate (PMMA) or similar materials.

Table 1 gives several examples of recent research reports relating to nanoparticle-mediated combined chemotherapy [15Yi X, Lian X, Dong J, et al. Co-delivery of Pirarubicin and Paclitaxel by human serum albumin nanoparticles to enhance antitumor effect and reduce systemic toxicity in breast cancers. Mol Pharm 2015; 12(11): 4085-98.
[http://dx.doi.org/10.1021/acs.molpharmaceut.5b00536] [PMID: 26422373] , 19Pradhan R, Ramasamy T, Choi JY, et al. Hyaluronic acid-decorated poly(lactic-co-glycolic acid) nanoparticles for combined delivery of docetaxel and tanespimycin. Carbohydr Polym 2015; 123: 313-23.
[http://dx.doi.org/10.1016/j.carbpol.2015.01.064] [PMID: 25843864] -25Jia L, Li Z, Shen J, et al. Multifunctional mesoporous silica nanoparticles mediated co-delivery of paclitaxel and tetrandrine for overcoming multidrug resistance. Int J Pharm 2015; 489(1-2): 318-30.
[http://dx.doi.org/10.1016/j.ijpharm.2015.05.010] [PMID: 25956050] , 27Zhang H, Tian Y, Zhu Z, et al. Efficient antitumor effect of co-drug-loaded nanoparticles with gelatin hydrogel by local implantation. Sci Rep 2016; 6: 26546.
[http://dx.doi.org/10.1038/srep26546] [PMID: 27226240] -30Li X, Lu X, Xu H, et al. Paclitaxel/tetrandrine coloaded nanoparticles effectively promote the apoptosis of gastric cancer cells based on oxidation therapy. Mol Pharm 2012; 9(2): 222-9.
[http://dx.doi.org/10.1021/mp2002736] [PMID: 22171565] ]. For instance, co-delivery of docetaxel (DTX) and tanespimycin (17-AAG) by hyaluronic acid (HA)-modified PLGA nanoparticles had shown the highest synergistic effect in killing MCF-7, MDA-MB-231 and SCC-7 cells at the molar ratio of 2:1 (DTX:17-AGG), and this synergistic antitumor activity was also demonstrated in vivo [19Pradhan R, Ramasamy T, Choi JY, et al. Hyaluronic acid-decorated poly(lactic-co-glycolic acid) nanoparticles for combined delivery of docetaxel and tanespimycin. Carbohydr Polym 2015; 123: 313-23.
[http://dx.doi.org/10.1016/j.carbpol.2015.01.064] [PMID: 25843864] ]. Furthermore, Wang et al. [20Wang B, Yu XC, Xu SF, Xu M. Paclitaxel and etoposide co-loaded polymeric nanoparticles for the effective combination therapy against human osteosarcoma. J Nanobiotechnology 2015; 13(1): 22.
[http://dx.doi.org/10.1186/s12951-015-0086-4] [PMID: 25880868] ] developed PEGylated PLGA nanoparticles for co-encapsulating paclitaxel (PTX) and etoposide (ETP) and demonstrated that co-delivery system enhanced cell cycle arrest and apoptosis of the human osteosarcom cells and improved cytotoxic activity of chemotherapeutic drugs.

Table 1
Nanoparticle-mediated combined chemotherapy.

PEG-PLGA and PEG-PLA nanoparticles have also been chemically altered to improve tumor targeting. For example, He et al. [21He Z, Huang J, Xu Y, et al. Co-delivery of cisplatin and paclitaxel by folic acid conjugated amphiphilic PEG-PLGA copolymer nanoparticles for the treatment of non-small lung cancer. Oncotarget 2015; 6(39): 42150-68.
[PMID: 26517524] , 22He Z, Shi Z, Sun W, et al. Hemocompatibility of folic-acid-conjugated amphiphilic PEG-PLGA copolymer nanoparticles for co-delivery of cisplatin and paclitaxel: treatment effects for non-small-cell lung cancer. Tumour Biol 2016; 37(6): 7809-21.
[http://dx.doi.org/10.1007/s13277-015-4634-1] [PMID: 26695149] ] utilized folic acid (FA)-modified PEG-PLGA nanoparticles for the co-delivery of cisplatin (CDDP) and PTX. This approach inhibited non-small cell lung cancer with a drug concentration ratio of 1:2 (CDDP:PTX). Similarly, Zhang [23Zhang X, Li J, Yan M. Targeted hepatocellular carcinoma therapy: transferrin modified, self-assembled polymeric nanomedicine for co-delivery of cisplatin and doxorubicin. Drug Dev Ind Pharm 2016; 42(10): 1590-9.
[http://dx.doi.org/10.3109/03639045.2016.1160103] [PMID: 26942448] ] and colleagues designed transferrin-modified PEG-PLGA nanoparticles (Tf-DDP/ DOX NPs) for co-delivery of CDDP and doxorubicin (DOX). Compared with free drugs, Tf-DDP/DOX NPs demonstrated greater antitumor activity for hepatoma carcinoma both in vivo and in vitro. Finally, Zhang et al. [24Zhu J, Xu X, Hu M, Qiu L. Co-encapsulation of combretastatin-A4 phosphate and doxorubicin in polymersomes for synergistic therapy of nasopharyngeal epidermal carcinoma. J Biomed Nanotechnol 2015; 11(6): 997-1006.
[http://dx.doi.org/10.1166/jbn.2015.2010] [PMID: 26353589] ] synthesized methoxy poly (ethylene glycol)-b-polylactide (mPEG-PLA) diblock copolymers by ring-opening polymerization. Combretastatin-A4 phosphate (CA4P) and DOX were loaded onto these mPEG-PLA nanoparticles, and demonstrated effective synergistic cytotoxicity, inhibiting human nasopharyngeal carcinoma angiogenesis and preventing proliferation of human nasopharyngeal epithelial cancer cells when the drug ratio of DOX:CA4P was 1:10.

In addition to using polymeric materials, inorganic materials, ceramides, and human serum albumin can also be used as nanocarriers. Human serum albumin (HSA), a protein found in the human serum, is particularly suited as a nanocarrier material due to its non-toxicity, biocompatible, and biodegradable [26Kratz F. Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release 2008; 132(3): 171-83.
[http://dx.doi.org/10.1016/j.jconrel.2008.05.010] [PMID: 18582981] ]. Yi et al. [15Yi X, Lian X, Dong J, et al. Co-delivery of Pirarubicin and Paclitaxel by human serum albumin nanoparticles to enhance antitumor effect and reduce systemic toxicity in breast cancers. Mol Pharm 2015; 12(11): 4085-98.
[http://dx.doi.org/10.1021/acs.molpharmaceut.5b00536] [PMID: 26422373] ] prepared a co-delivery system of PTX and pirarubicin (THP) by using HSA, and their results indicated that the nanoparticles had greater cytotoxicity against 4T1 breast cancer cells when compared with single or free drug delivery. Furthermore, the side effects of the drugs were significantly reduced, including bone marrow suppression, and organ or gastrointestinal toxicity.

Liposomes are spherical envelopes with a lipoid bilayer, adapted to encapsulate drugs [31Tardi P, Johnstone S, Harasym N, et al. In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy. Leuk Res 2009; 33(1): 129-39.
[http://dx.doi.org/10.1016/j.leukres.2008.06.028] [PMID: 18676016] ]. When compared with nanoparticles, liposomes have greater biocompatibility, and some liposome-mediated combined chemotherapy preparations have entered into clinical trials. There searchers at Celator Pharmaceuticals utilized distearoylphosphatidylglycerol (DSPG), distearoylphosphatidylcholine (DSPC), and cholesterol (Chol) to prepare liposomes for co-encapsulating cytarabine (Ara-C) and daunorubicin (DNR) at a molar ratio of 5:1. The resulting drug, CPX-351, significantly inhibited leukemia cells in vitro (while maintaining tumor cell selectivity), and the two drugs still acted synergistically in vivo, with similar anti-leukemia effects [31Tardi P, Johnstone S, Harasym N, et al. In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy. Leuk Res 2009; 33(1): 129-39.
[http://dx.doi.org/10.1016/j.leukres.2008.06.028] [PMID: 18676016] -33Lim WS, Tardi PG, Dos Santos N, et al. Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts. Leuk Res 2010; 34(9): 1214-23.
[http://dx.doi.org/10.1016/j.leukres.2010.01.015] [PMID: 20138667] ]. Furthermore, CPX-351 is in stage III of clinical trials, and the latest reports indicate that the US FDA has granted breakthrough therapy status to Celator Pharmaceuticals’ CPX-351 for the treatment of adults with therapy-related acute myeloid leukemia (t-AML) and AML with myelodysplasia-related changes (AML-MRC).

A list of recent studies concerning liposome-mediated combined chemotherapy is presented in Table 2 [34Riviere K, Kieler-Ferguson HM, Jerger K, Szoka FC Jr. Anti-tumor activity of liposome encapsulated fluoroorotic acid as a single agent and in combination with liposome irinotecan. J Control Release 2011; 153(3): 288-96.
[http://dx.doi.org/10.1016/j.jconrel.2011.05.005] [PMID: 21600250] -44Wu M, Fan Y, Lv S, et al. Vincristine and Temozolomide combined chemotherapy for the treatment of glioma: a comparison of solid lipid nanoparticles and nanostructured lipid carriers for dual drugs delivery. Drug Del 2015; pp. 1-6.]. For example, Riviereet et al. [34Riviere K, Kieler-Ferguson HM, Jerger K, Szoka FC Jr. Anti-tumor activity of liposome encapsulated fluoroorotic acid as a single agent and in combination with liposome irinotecan. J Control Release 2011; 153(3): 288-96.
[http://dx.doi.org/10.1016/j.jconrel.2011.05.005] [PMID: 21600250] ] compared the antitumor effect of co-delivery of the synergistic drugs fluoroorotic acid (FOA) and irinotecan (IRN) in the same liposome versus delivery of the same drugs in separate liposomes. The co-delivery system was more successful in inhibiting the proliferation of C26 cells in vitro; however, the drugs did not have synergistic effects in the C26 tumor mouse model [34Riviere K, Kieler-Ferguson HM, Jerger K, Szoka FC Jr. Anti-tumor activity of liposome encapsulated fluoroorotic acid as a single agent and in combination with liposome irinotecan. J Control Release 2011; 153(3): 288-96.
[http://dx.doi.org/10.1016/j.jconrel.2011.05.005] [PMID: 21600250] ]. These results demonstrate the challenges associated with developing synergistic treatment protocols in vivo based on in vitro studies.

Table 2
Liposome-mediated combined chemotherapy.

Our group reported an effective strategy for targeting liver cancer cells (HepG2) and liver cancer stem cells (HepG2-TS) by developing liposomes for co-delivery of salinomycin (SAL) and chloroquine (CQ) [35Xie F, Zhang S, Liu J, et al. Codelivery of salinomycin and chloroquine by liposomes enables synergistic antitumor activity in vitro. Nanomedicine (Lond) 2016; 11(14): 1831-46.
[http://dx.doi.org/10.2217/nnm-2016-0125] [PMID: 27391366] ]. After screening, the synergistic molar ratio of SAL:CQ was 1:5, the liposome particle size was about 120nm, and the PDI was greater. In vitro cytology results showed that combining drugs could improve the cytotoxic effect of SAL in HepG2 cells, but not in HepG2-TS. We surmised that the mechanism might be related to the basal autophagy flux, as autophagy plays a protective role in liver cancer. Therefore, we measured the basal autophagy flux of the two cell types, and demonstrated that it was significantly greater in HepG2 cells. When SAL was combined with CQ in HepG2 cells,the antitumor effect of SAL was improved because CQ could inhibit autophagy [35Xie F, Zhang S, Liu J, et al. Codelivery of salinomycin and chloroquine by liposomes enables synergistic antitumor activity in vitro. Nanomedicine (Lond) 2016; 11(14): 1831-46.
[http://dx.doi.org/10.2217/nnm-2016-0125] [PMID: 27391366] ]. Furthermore, our group loaded SAL and DOX into liposomes separately, and in combination, to assess the subsequent antitumor activity in liver cancer. The results showed that co-delivery liposomes and separate liposomes had similar antitumor activity [36Gong Z, Chen D, Xie F, et al. Codelivery of salinomycin and doxorubicin using nanoliposomes for targeting both liver cancer cells and cancer stem cells. Nanomedicine (Lond) 2016; 11(19): 2565-79.
[http://dx.doi.org/10.2217/nnm-2016-0137] [PMID: 27647449] ].

In addition, Morton et al. used the chemical properties of liposomes and drugs to create a controlled sequence of drug release [37Morton SW, Lee MJ, Deng ZJ, et al. A nanoparticle-based combination chemotherapy delivery system for enhanced tumor killing by dynamic rewiring of signaling pathways. Sci Signal 2014; 7(325): ra44.
[http://dx.doi.org/10.1126/scisignal.2005261] [PMID: 24825919] ]. The hydrophobic drug erlotinib and the hydrophilic drug DOX were encapsulated in the hydrophobic compartment and the hydrophilic interior of liposomes, respectively. This enabled them to achieve the desired time sequence of drug release in which erlotinib first inhibited EGFR activity in cancer cells and changed the intracellular apoptotic pathways, thus improving the sensitivity of breast cancer cells to the later release of DOX. Encouragingly, this time-staggered drug delivery could potentially enhance antitumor effects in vivo [37Morton SW, Lee MJ, Deng ZJ, et al. A nanoparticle-based combination chemotherapy delivery system for enhanced tumor killing by dynamic rewiring of signaling pathways. Sci Signal 2014; 7(325): ra44.
[http://dx.doi.org/10.1126/scisignal.2005261] [PMID: 24825919] ].

Lipid-polymer hybrid nanoparticles are prepared using polymeric and lipid materials which exhibit a core-shell structure [45Hadinoto K, Sundaresan A, Cheow WS. Lipid-polymer hybrid nanoparticles as a new generation therapeutic delivery platform: a review. Eur J Pharm Biopharm 2013; 85(3 Pt A): 427-43.
[http://dx.doi.org/10.1016/j.ejpb.2013.07.002] [PMID: 23872180] ]. They possess a wide range of potential applications, as they overcome some of the drawbacks associated with liposome nanoparticles: they are less physically and chemically unstable and they are easier to store, thus reducing drug leakage into the circulation. Furthermore, they have greater drug loading capacity than polymer nanoparticles, and exhibit better tumor targeting [45Hadinoto K, Sundaresan A, Cheow WS. Lipid-polymer hybrid nanoparticles as a new generation therapeutic delivery platform: a review. Eur J Pharm Biopharm 2013; 85(3 Pt A): 427-43.
[http://dx.doi.org/10.1016/j.ejpb.2013.07.002] [PMID: 23872180] -47Wu XY. Strategies for optimizing polymer-lipid hybrid nanoparticle-mediated drug delivery. Expert Opin Drug Deliv 2016; 13(5): 609-12.
[http://dx.doi.org/10.1517/17425247.2016.1165662] [PMID: 26978527] ]. The recent research regarding lipid-polymer hybrid nanoparticle-mediated combined chemotherapy is summarized in Table 3 [48Zhao X, Chen Q, Li Y, Tang H, Liu W, Yang X. Doxorubicin and curcumin co-delivery by lipid nanoparticles for enhanced treatment of diethylnitrosamine-induced hepatocellular carcinoma in mice. Eur J Pharm Biopharm 2015; 93: 27-36.
[http://dx.doi.org/10.1016/j.ejpb.2015.03.003] [PMID: 25770771] -52Wang C, Su L, Wu C, Wu J, Zhu C, Yuan G. RGD peptide targeted lipid-coated nanoparticles for combinatorial delivery of sorafenib and quercetin against hepatocellular carcinoma. Drug Dev Ind Pharm 2016; 42(12): 1938-44.
[http://dx.doi.org/10.1080/03639045.2016.1185435] [PMID: 27142812] ].

Table 3
Lipid-polymer hybrid nanoparticle-mediated combined chemotherapy.

Of particular note, Zhao [48Zhao X, Chen Q, Li Y, Tang H, Liu W, Yang X. Doxorubicin and curcumin co-delivery by lipid nanoparticles for enhanced treatment of diethylnitrosamine-induced hepatocellular carcinoma in mice. Eur J Pharm Biopharm 2015; 93: 27-36.
[http://dx.doi.org/10.1016/j.ejpb.2015.03.003] [PMID: 25770771] ] and colleagues employed lipid-polymer hybrid nanoparticles to encapsulate curcumin (CUR) and DOX to investigate their antitumor activity on diethylnitrosamine (DEN)-induced hepatocellular carcinoma in mice. The results showed that co-delivery of CUR and DOX in this way enhanced their antitumor effects, promoting apoptosis of tumor cells and inhibiting both tumor cell proliferation and angiogenesis. Further analysis also indicated that DOX/CUR-NPs had greater antitumor effects than DOX-NPs, which was related to regulation of the levels of hypoxia-associated mRNA and protein, as well as drug resistance [48Zhao X, Chen Q, Li Y, Tang H, Liu W, Yang X. Doxorubicin and curcumin co-delivery by lipid nanoparticles for enhanced treatment of diethylnitrosamine-induced hepatocellular carcinoma in mice. Eur J Pharm Biopharm 2015; 93: 27-36.
[http://dx.doi.org/10.1016/j.ejpb.2015.03.003] [PMID: 25770771] ]. In another study, Zhang et al. [49Zhang J, Hu J, Chan HF, Skibba M, Liang G, Chen M. iRGD decorated lipid-polymer hybrid nanoparticles for targeted co-delivery of doxorubicin and sorafenib to enhance anti-hepatocellular carcinoma efficacy. Nanomedicine (Lond) 2016; 12(5): 1303-11.
[PMID: 26964482] ] successfully bound iRGD peptide to lipid-polymer hybrid nanoparticles in order to enhance the antitumor efficacy of DOX and sorafenib (SOR). Compared with the free drugs and nanoparticles without iRGD peptide modification, DOX+SOR/iRGD-NPs had greater synergistic cytotoxic effects; they also prolonged the cycle time, improved the biocompatibility, and enhanced endocytosis and drug accumulation in the tumor. In a mouse model of liver cancer, the tumor inhibition effect of these DOX+SOR/iRGD-NPs was even more significant [49Zhang J, Hu J, Chan HF, Skibba M, Liang G, Chen M. iRGD decorated lipid-polymer hybrid nanoparticles for targeted co-delivery of doxorubicin and sorafenib to enhance anti-hepatocellular carcinoma efficacy. Nanomedicine (Lond) 2016; 12(5): 1303-11.
[PMID: 26964482] ].

Furthermore, although lipid-polymer hybrid nanoparticles are generally prepared using polymeric and lipid materials, they have also been produced from inorganic materials and liposomes. In a recent research by Choi et al. [50Choi JY, Ramasamy T, Kim SY, et al. PEGylated lipid bilayer-supported mesoporous silica nanoparticle composite for synergistic co-delivery of axitinib and celastrol in multi-targeted cancer therapy. Acta Biomater 2016; 39: 94-105.
[http://dx.doi.org/10.1016/j.actbio.2016.05.012] [PMID: 27163403] ], combined ACML nanoparticles were formed by loading axitinib (AXT) in PEGylated lipid bilayers and celastrol (CST) in MSN. Cytological experiments demonstrated that ACML could inhibit both angiogenesis and mitochondrial function and induce apoptosis in several cell types (SCC-7, BT-474 breast cancer cells and SH-SY5Y neuroblastoma cells). Notably, the tumor inhibition effects were even more significant in mouse tumor models.

Another promising form of nanocarrier is the polymer micelle, and several drug-loaded micelles have entered into clinical trials (for example, the PTX-loaded polymer micelles, NK105, and the CDDP-loaded polymer micelles, NC-6004) [53Cabral H, Kataoka K. Progress of drug-loaded polymeric micelles into clinical studies. J Control Release 2014; 190: 465-76.
[http://dx.doi.org/10.1016/j.jconrel.2014.06.042] [PMID: 24993430] -55Plummer R, Wilson RH, Calvert H, et al. A Phase I clinical study of cisplatin-incorporated polymeric micelles (NC-6004) in patients with solid tumours. Br J Cancer 2011; 104(4): 593-8.
[http://dx.doi.org/10.1038/bjc.2011.6] [PMID: 21285987] ]. Table 4 provides an overview of recent studies using polymer micelle-mediated combined chemotherapy [56Desale SS, Cohen SM, Zhao Y, Kabanov AV, Bronich TK. Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer. J Control Release 2013; 171(3): 339-48.
[http://dx.doi.org/10.1016/j.jconrel.2013.04.026] [PMID: 23665258] -62Abouzeid AH, Patel NR, Torchilin VP. Polyethylene glycol-phosphatidylethanolamine (PEG-PE)/vitamin E micelles for co-delivery of paclitaxel and curcumin to overcome multi-drug resistance in ovarian cancer. Int J Pharm 2014; 464(1-2): 178-84.
[http://dx.doi.org/10.1016/j.ijpharm.2014.01.009] [PMID: 24440402] ].

Table 4
Polymer micelle-mediated combined chemotherapy.

In order to eradicate ovarian cancer, Desale et al. [56Desale SS, Cohen SM, Zhao Y, Kabanov AV, Bronich TK. Biodegradable hybrid polymer micelles for combination drug therapy in ovarian cancer. J Control Release 2013; 171(3): 339-48.
[http://dx.doi.org/10.1016/j.jconrel.2013.04.026] [PMID: 23665258] ] synthesized biodegradable triblock copolymers containing ethylene glycol, glutamic acid, and phenylalanine (PEG–PGlu–PPhe). These copolymers self-assembled to form micelles for the co-delivery of CDDP and PTX, and the resulting nanomedicine mounted strong tumor suppression effects both in vitro and in vivo. Similarly, Cai et al. [57Cai L, Xu G, Shi C, Guo D, Wang X, Luo J. Telodendrimer nanocarrier for co-delivery of paclitaxel and cisplatin: A synergistic combination nanotherapy for ovarian cancer treatment. Biomaterials 2015; 37: 456-68.
[http://dx.doi.org/10.1016/j.biomaterials.2014.10.044] [PMID: 25453973] ]. used polymeric micelles containing CDDP and PTX for the treatment of ovarian cancer. They developed three-layered linear-dendritic telodendrimer micelles and loaded drugs at a 2:1 molar ratio of CDDP:PTX. Compared with the free drug combination and loading in separate micelles, the co-delivery micelles provided greater tumor targeting and prolonged drug half-life. Moreover, the antitumor effect was significant and it could enhance the survival of mice [57Cai L, Xu G, Shi C, Guo D, Wang X, Luo J. Telodendrimer nanocarrier for co-delivery of paclitaxel and cisplatin: A synergistic combination nanotherapy for ovarian cancer treatment. Biomaterials 2015; 37: 456-68.
[http://dx.doi.org/10.1016/j.biomaterials.2014.10.044] [PMID: 25453973] ].

Finally, Duan et al. [58Duan X, Xiao J, Yin Q, et al. Smart pH-sensitive and temporal-controlled polymeric micelles for effective combination therapy of doxorubicin and disulfiram. ACS Nano 2013; 7(7): 5858-69.
[http://dx.doi.org/10.1021/nn4010796] [PMID: 23734880] ] developed a pH-sensitive polymeric micelle system to achieve the temporal release of two drugs. The researchers conjugated DOX to a poly (styrene-co-maleic anhydride) (SMA) derivative with adipic dihydrazide (ADH) through an acid-cleavable hydrazone bond. They then loaded disulfiram (DSF) into the polymer micelles through the self-assembly of a SMA-ADH-DOX (SAD) conjugate [58Duan X, Xiao J, Yin Q, et al. Smart pH-sensitive and temporal-controlled polymeric micelles for effective combination therapy of doxorubicin and disulfiram. ACS Nano 2013; 7(7): 5858-69.
[http://dx.doi.org/10.1021/nn4010796] [PMID: 23734880] ]. This nanomedicine rapidly released DSF to inhibit the activity of P-glycoprotein and then slowly released DOX to increase its accumulation within tumor cells and enhance the antitumor response.

Apart from the previously mentioned nanocarriers, microemulsions and microspheres are also traditional drug carriers. Owing to the relatively large particle size, these drug carriers have some unique properties such as a high drug-loading capacity and reduced drug leakage. Noh et al. [63Noh I, Kim HO, Choi J, et al. Co-delivery of paclitaxel and gemcitabine via CD44-targeting nanocarriers as a prodrug with synergistic antitumor activity against human biliary cancer. Biomaterials 2015; 53: 763-74.
[http://dx.doi.org/10.1016/j.biomaterials.2015.03.006] [PMID: 25890771] ] designed and prepared multi-prodrug nanocarriers (MPDNCs), with PTX being conjugated with polylysine carboxylate to form a cationic polymer (PLL-PTX), and HA and gemcitabine (GEM) combined to form an anionic polymer (HA-GEM). The two types of polymer then formed the multi-prodrug nanocarrier through electrostatic interaction. Because HA binds to the CD44 receptor, these MPDNCs targeted the CD44 over expression of the biliary cancer cell lines, HuCCT1, and therefore markedly inhibited cancer cell proliferation. Furthermore, they also induced cancer cell apoptosis and improved anti-tumor activity [63Noh I, Kim HO, Choi J, et al. Co-delivery of paclitaxel and gemcitabine via CD44-targeting nanocarriers as a prodrug with synergistic antitumor activity against human biliary cancer. Biomaterials 2015; 53: 763-74.
[http://dx.doi.org/10.1016/j.biomaterials.2015.03.006] [PMID: 25890771] ].

Similarly, Lee et al. [64Lee WL, Guo WM, Ho VH, et al. Delivery of doxorubicin and paclitaxel from double-layered microparticles: The effects of layer thickness and dual-drug vs. single-drug loading. Acta Biomater 2015; 27: 53-65.
[http://dx.doi.org/10.1016/j.actbio.2015.08.051] [PMID: 26340886] ] prepared double-layered microparticles using DOX loaded into the PLGA shell and PTX loaded into the poly (L-lactic acid) (PLLA) core. By changing the polymer ratio, the burst release of DOX was controlled and the drug release time prolonged to 2 months. The double-layered microparticles significantly reduced the rate of formation of microspheres and had a good antitumor effect when incubated with MCF-7 microspheres [64Lee WL, Guo WM, Ho VH, et al. Delivery of doxorubicin and paclitaxel from double-layered microparticles: The effects of layer thickness and dual-drug vs. single-drug loading. Acta Biomater 2015; 27: 53-65.
[http://dx.doi.org/10.1016/j.actbio.2015.08.051] [PMID: 26340886] ].

As discussed above, co-delivery of chemotherapeutic agents in nanoparticles can improve the effects of combined chemotherapy for the treatment of tumors. However, there is also evidence that loading single drugs into separate nanoparticles can also achieve the desired antitumor effect. These preparations overcome certain difficulties associated with co-delivery, namely that the drug combinations are flexible, the synergistic ratio of the drugs is maintained, and drugs with disparate chemical properties can be effectively delivered within their own uniquely tailored carrier particles. Nevertheless, it is difficult to control the synergistic ratio and the concentration of the two drugs at the target site in vivo, so the success of this strategy is dependent upon the specific circumstances in which it is used.

In general, two kinds of chemotherapeutic drugs are respectively loaded into the carriers to target both cancer stem cells and tumor cells. Cancer stem cells are a form of undifferentiated or primitive cells, with the capacity for multipotent differentiation and self-renewal ability [65Song H, Su X, Yang K, et al. CD20 Antibody-Conjugated immunoliposomes for targeted chemotherapy of melanoma cancer initiating cells. J Biomed Nanotechnol 2015; 11(11): 1927-46.
[http://dx.doi.org/10.1166/jbn.2015.2129] [PMID: 26554153] -69Yin C, Xie WF. Differentiation therapy with transcription factors might present as an ideal strategy for the treatment of cancer. Hepatology 2009; 50(6): 2046-7.
[http://dx.doi.org/10.1002/hep.23328] [PMID: 19937684] ]. Our group has used this strategy previously to target MCF-7 and MCF-7 microspheres [70Wang D, Huang J, Wang X, et al. The eradication of breast cancer cells and stem cells by 8-hydroxyquinoline-loaded hyaluronan modified mesoporous silica nanoparticle-supported lipid bilayers containing docetaxel. Biomaterials 2013; 34(31): 7662-73.
[http://dx.doi.org/10.1016/j.biomaterials.2013.06.042] [PMID: 23859657] ]. In this study, we used silane coupling agent-modified MSN to separately encapsulate 8-hydroxyquinoline (8-HQ) and DTX. The DTX-loaded MSN and 8-HQ-loaded MSN were then covered with lipid bilayers and HA-bound lipid bilayers, respectively, producing DTX-MSS and 8-HQ-HA-MSS. Our results demonstrated that DTX-MSS was more effective in inhibiting MCF-7 than MCF-7 microspheres, whereas the role of 8-HQ-HA-MSS was just the opposite . Therefore, combining the two forms of nanomedicine resulted in better antitumor activity and reduced drug toxicity in normal cells [70Wang D, Huang J, Wang X, et al. The eradication of breast cancer cells and stem cells by 8-hydroxyquinoline-loaded hyaluronan modified mesoporous silica nanoparticle-supported lipid bilayers containing docetaxel. Biomaterials 2013; 34(31): 7662-73.
[http://dx.doi.org/10.1016/j.biomaterials.2013.06.042] [PMID: 23859657] ].

In another example of this approach, Ke et al. [71Ke XY, Lin Ng VW, Gao SJ, Tong YW, Hedrick JL, Yang YY. Co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells. Biomaterials 2014; 35(3): 1096-108.
[http://dx.doi.org/10.1016/j.biomaterials.2013.10.049] [PMID: 24183698] ] used polymer micelles to target tumor cells and cancer stem cells. The polymer micelles were prepared using two diblock copolymers, one of PEG and urea-functionalized polycarbonate (PEG-PUC), and another of PEG and acid-functionalized polycarbonate (PEG-PAC) in a 1:1 molar ratio. Thioridazine (THZ) and DOX were loaded into these polymer micelles, respectively (THZ-MM and DOX-MM). It has been reported that THZ could kill cancer stem cells [72Sachlos E, Risueño RM, Laronde S, et al. Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells. Cell 2012; 149(6): 1284-97.
[http://dx.doi.org/10.1016/j.cell.2012.03.049] [PMID: 22632761] ].The results confirmed that the free drug THZ and THZ-MM had inhibitory effect on cancer stem cells, and both the free drugs in combination and the polymer micelle combination could effectively inhibit the proliferation of cancer stem cells. However, the polymer micelle combination was found to have the most significant antitumor effect in a BT-474 nude mice tumor model [71Ke XY, Lin Ng VW, Gao SJ, Tong YW, Hedrick JL, Yang YY. Co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells. Biomaterials 2014; 35(3): 1096-108.
[http://dx.doi.org/10.1016/j.biomaterials.2013.10.049] [PMID: 24183698] ].

Finally, Li et al. [73Li SY, Sun R, Wang HX, et al. Combination therapy with epigenetic-targeted and chemotherapeutic drugs delivered by nanoparticles to enhance the chemotherapy response and overcome resistance by breast cancer stem cells. J Control Release 2015; 205: 7-14.
[http://dx.doi.org/10.1016/j.jconrel.2014.11.011] [PMID: 25445694] ] demonstrated that decitabine (DAC) and DOX-loaded nanoparticles had the best inhibitory effect on cancer stem cells.

Cancer is one of the main threats to human health; therefore, the question of how to cure it has become an important research topic worldwide. Currently, chemotherapy is a common mode of treatment, and the combination of two or more chemotherapeutic agents greatly improves the therapeutic effect. However, this combined strategy has significant drawbacks, namely drug toxicity and difficulty maintaining the synergistic ratio of the drugs, which leads to treatment failure. Nanomedicine may provide a solution for this problem by increasing the antitumor activity of combined chemotherapy through novel delivery methods. At present, however, few preparations progress to the clinical research stage, and the scientific community faces challenges in terms of enhancing the antitumor effects, overcoming practical application problems, and expanding the scope of nanomedicine-mediated combined chemotherapy. We believe that with scientific and technological progress, these challenges are not insurmountable and that nanomedicine-mediated combined chemotherapy will succeed in the treatment of cancer.