Application of Nanocarriers in Co-Loading siRNA and Chemotherapeutic Drugs to Reverse Multidrug Resistance of Tumor

Qiangqiang Yang; Chuan Li; Shuxian Yu; Shuhua Fan; Yuexia Wang; Min Hong

doi:10.7536/PC200956

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (10) : 1900-1916 DOI: 10.7536/PC200956

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Application of Nanocarriers in Co-Loading siRNA and Chemotherapeutic Drugs to Reverse Multidrug Resistance of Tumor

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Abstract

In recent years, gene interference technology based on small interfering RNA(siRNA) has shown great potential in reversing multidrug resistance of chemotherapy by regulating various proteins related to multidrug resistance of tumor at the gene level. In view of this, the researchers have done a lot of work in the area of RNA interference and chemotherapy drugs. But the free siRNA is not easy to be absorbed by cells without carrier, and it will be degraded by endogenous ribonuclease in plasma and tissues. Therefore, it is necessary to load siRNA on the carrier in order to effectively apply it to tumor treatment. In view of the safety, high efficiency and targeting of nanocarriers, a large number of nanocomposite systems have been developed which can simultaneously load siRNA and chemotherapy drugs. In this paper, the application of nanomaterials in co-loading siRNA and reversing multidrug resistance of chemotherapy drugs are reviewed. In addition, in order to have a better understanding of the related work, some targets which are often used in the study to reverse multidrug resistance are also briefly described.

Contents

1 Introduction

2 Main targets of siRNA regulation

2.1 ATP-binding cassette transporters

2.2 Apoptosis related proteins

2.3 Other targets

3 Nanocarriers for co-delivery of siRNA and chemotherapeutic drugs

3.1 Mesoporous silica nanoparticles

3.2 Metal-organic frameworks

3.3 Polymeric micelles

3.4 Liposome/Niosome

3.5 Dendrimer

3.6 layer-by-Layer nanoparticles

3.7 Hyaluronic acid nanosystem

3.8 Summary

4 Expansion

5 Conclusion and outlook

Graphical abstract

Keywords

RNA interference technology / gene regulation / reversal of multidrug resistance / nanocarrier

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Qiangqiang Yang, Chuan Li, Shuxian Yu, Shuhua Fan, Yuexia Wang, Min Hong. Application of Nanocarriers in Co-Loading siRNA and Chemotherapeutic Drugs to Reverse Multidrug Resistance of Tumor. Progress in Chemistry, 2021, 33(10): 1900-1916 DOI:10.7536/PC200956

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1 Introduction

According to the report of the International Cancer Research Institute in 2018, the proportion of malignant tumors in the world population has increased year by year since 2010. The American Cancer Society estimates that drug resistance affects more than 90% of cancer deaths to some degree. multi-drug resistance (MDR) refers to the cross-resistance of tumor cells to other anti-tumor drugs with different structures and mechanisms of action while they are resistant to one anti-tumor drug, also known as multidrug resistance^[1].

Usually, when drugs play a role, the complex microenvironment of tumor tissue itself will bring great obstacles to carrier-free chemotherapy drugs or gene drugs. For example, the microenvironment of tumor tissue, such as abnormal vascular structure and rigid extracellular matrix, can cause difficulties in drug delivery in tumor tissue. Hypoxic tumor-specific physiological parameters can reduce the response of tumor cells to drugs. Acidic pH physiological parameters can upregulate angiogenic factors and proteases, and promote drug resistance. Stromal cells in the vicinity of tumor cells secrete a variety of factors to modify the tumor matrix, thus promoting the occurrence, development, metastasis and drug resistance of tumors. Cancer stem cells highly express ABC transporter and have the ability of self-renewal, which is one of the main reasons for drug resistance and recurrence of tumors^[2].

In a word, the above are the reasons for the poor efficacy and drug resistance of tumor drugs at the level of tumor tissues, and the study of drug resistance at the level of cells is related to many factors in cells.For example, (1) ATP-binding cassette (ABC) transporters with efflux pump function are highly expressed in tumor cells, which are related to reducing the uptake of chemotherapeutic drugs, promoting drug efflux and reducing the intracellular drug content.For example, high expression of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP)^[3⇓⇓~6]; (2) Tumor cells are resistant to apoptosis. Most chemotherapeutic drugs kill tumor cells by inducing apoptosis, and the overexpression of apoptosis-related proteins such as Bcl-2, Mcl-1 and PLK1 is related to tumor activation and anti-apoptosis^[7⇓~9]. (3) Increased drug metabolism, such as increased expression and activity of glutathione S-transferase (GST), will increase the metabolism of chemotherapeutic drugs and reduce the concentration of drugs in cells^[10]. In addition, recent studies have shown that tumor multidrug resistance is also related to the high expression of DNA repair and replication-related enzymes such as O6-methylguanine DNA-methyltransferase (MGMT) and ribonucleic acid reductase M2 (RRM2)^[11].

Gene therapy has proved to be a very promising anti-cancer method in clinic. In gene therapy, gene interference is usually used to shut down or inhibit the expression of pathogenic genes in order to achieve the purpose of cancer treatment. At present, the main methods of gene interference are antisense oligonucleotide technology, ribozyme and deoxyribozyme technology, RNA interference technology, microRNA technology and so on. RNA interference (RNAi) is a gene regulation method, which is mediated by short double-stranded small interference RNA (siRNA) to degrade homologous mRNA molecules and inhibit the expression of related genes. In recent years, siRNA-based RNA interference technology has developed rapidly in the treatment of cancer. Besides the anti-cancer effect of siRNA drugs has been confirmed in cancer animal models, some of them have entered the clinical trial stage and been used in the treatment of solid tumors^{[12⇓⇓⇓~16]}^[17]. However, intravenous injection of carrier-free siRNA is not ideal for the treatment of tumors. Free siRNA is a hydrophilic anionic siRNA, which is not easily absorbed by cells in the absence of a carrier, and the existence of endogenous ribonuclease (RNase) in plasma and tissues makes the half-life of free siRNA very short, while renal clearance and phagocyte uptake lead to lower levels of free siRNA in vivo^[18]^[19,20]^[21,22]. Therefore, siRNA must be loaded on the vector in order to be effectively used in cancer therapy. Nanocarriers have the advantages of safety, high efficiency and targeting, and siRNA is encapsulated on nanomaterials before administration in clinic^[23].

Chemotherapy is the main method of cancer treatment in clinic. The main problems of free chemotherapeutic drugs are as follows: (1) Most chemotherapeutic drugs have poor water solubility^[24]; (2) After administration, the drug will be non-specifically distributed throughout the body, but the permeability at the tumor site is insufficient^[25]; (3) its toxicity to normal tissues limits its therapeutic dose and frequency of administration; (4) It is easy to produce drug resistance. In view of the above problems, researchers consider loading chemotherapeutic drugs on nano-carriers to increase the solubility of drugs, and at the same time, using the EPR effect of tumors to improve the permeability and retention of chemotherapeutic drugs in tumor tissues.It can reduce the damage to normal tissues, break through the original dosage, change the distribution of drugs in organelles (such as nuclear targeting), and increase the concentration of drugs in target organelles. When tumor cells are repeatedly exposed to chemotherapeutic drugs, defense proteins will be produced in the cytoplasm to inactivate the drugs, but the application of nano-carriers to load chemotherapeutic drugs can bypass proteins such as P-gp, which are highly expressed in cell membranes and nuclear membranes and have the function of efflux drugs.As well as defense proteins produced in the cytoplasm, the reason may be to overcome drug resistance by bypassing the cell's visual defense system and producing a Trojan horse effect^[26,27]. Therefore, the loading of chemotherapeutic drugs on nanocarriers is of great significance to overcome the resistance of chemotherapeutic drugs^[28⇓~30]. Paclitaxel (PTX), cisplatin, Gemcitabine (Gem), Doxorubicin (DOX) and Docetaxel (DTX) are the most commonly used chemotherapeutic drugs. The existence of drug resistance promotes the development of chemotherapy from single drug to multi-line drug chemotherapy. For example, in the treatment of ovarian cancer, platinum drugs are generally chosen as the first chemotherapy drugs, except for patients who are resistant to platinum drugs.Even for platinum-sensitive patients, with the increase of drug use, impaired DNA repair and changes in apoptosis process, patients will inevitably develop drug resistance, followed by a variety of chemotherapeutic drugs.For example, platinum combined with Gem, platinum combined with PTX, but with the progress of treatment, there will be patients who are resistant to both platinum and PTX, so we need to continue to explore treatment options.Combination therapy with other chemotherapeutic agents, such as irinotecan in combination with apatinib for advanced ovarian cancer resistant to both platinum and taxanes, or with resistance-associated protein inhibitors^[31]^[32]^[33]^[34]. At present, there are more and more studies on the mechanism of drug resistance, but the specific drugs for these targets are limited, so it is necessary to find more effective treatment methods.

Nanomaterials are used as carriers to jointly deliver siRNA and chemotherapeutic drugs, which can not only produce Trojan horse drug delivery effect and improve the anti-cancer effect of chemotherapeutic drugs,At the same time, the loaded siRNA can block the expression of proteins related to multidrug resistance by degrading the abnormally expressed genes of drug-resistant cells, destroy the cellular pathways of drug-resistant cells, and reverse the drug-resistant environment of tumor cells^[35,36]. It can be seen that co-loading siRNA and chemotherapeutic drugs can play a synergistic role between different drugs, produce better anti-tumor effect, and have great application prospects in reversing drug resistance. In addition to co-loading siRNA and chemotherapeutic drugs on nano-carriers, further combining target molecules on nano-carriers to achieve active targeted drug delivery is also a research hotspot to overcome drug resistance, such as modifying cell penetrating peptides (CPPs) on the surface of nano-carriers, because CPPs can escape P-gp-mediated MDR, nano-drugs can enhance cell penetration and increase intracellular drug accumulation^[37,38].

At present, in the aspect of overcoming drug resistance, there have been studies in this field based on the co-loading of chemotherapeutic drugs and related inhibitors by nanocarriers, or the combination of chemotherapeutic drugs with pro-apoptotic agents.For example, chemotherapy drugs combined with P-gp inhibitors cyclosporine and verapamil, but these inhibitors have the problem that the toxic dose is similar to the therapeutic dose, which is easy to cause adverse reactions. Although the nanocarrier is targeted, it will inevitably be phagocytosed by the reticuloendothelial system (RES system) in vivo, such as liver, kidney and spleen, which shows that the co-delivery of related inhibitors or pro-apoptotic agents has higher toxicity compared with the co-delivery of siRNA^[39]. In addition, the mechanism of drug resistance in different types of cancer cells is not completely consistent, the drug-resistance-related proteins are diverse, the types of drugs are limited, and the proteins may not be specifically inhibited, but with the development of gene science, siRNA has versatility in inhibiting different drug-resistant proteins, can specifically down-regulate drug-resistant genes, and has a very broad application prospect.

In order to realize the combination therapy of siRNA and chemotherapeutic drugs on the basis of nano-carriers, separate administration and co-delivery of siRNA and chemotherapeutic drugs can be used, but the latter is obviously more advantageous. For example, co-delivery nanosystems have the potential to deliver drugs step by step, first releasing siRNA to down-regulate drug-resistance-related proteins, so that tumor cells are sensitive to chemotherapeutic drugs, and then chemotherapeutic drugs play a role to achieve the B est therapeutic effect; In addition, co-delivery administration can achieve the purpose of combination therapy only by single administration, which can improve patient compliance in terms of medication time and frequency.

In the related work of co-loading siRNA and chemotherapeutic drugs with nanocarriers, researchers basically choose some conventional chemotherapeutic drugs (such as DOX, PTX, DTX, cisplatin or Gem), but the target types of nanocarriers and siRNA used are diverse.In order to better discuss the work of researchers in co-loading siRNA and chemotherapeutic drugs on nanocarriers, we first summarize and briefly introduce the targets selected by researchers for RNA interference in recent years.Then, the achievements of researchers based on the concept of co-loading of nanocarriers in recent years are summarized according to different nanocarriers.

2 Regulatory Targets of RNA Interference Based on siRNA

In the process of cancer treatment, chemotherapy failure is often caused by drug resistance, and people have been trying to explore the mechanism of drug resistance, and gradually found that drug resistance is closely related to some genes and proteins abnormally expressed in cancer cells.These proteins are classified according to their functions, mainly including ATP-binding cassette transporters with efflux function, proteins with anti-apoptotic effect, and proteins and enzymes in some cellular pathways. Therefore, people choose the mRNA corresponding to these proteins as the target, and use siRNA-based RNA interference technology to down-regulate the expression of these targets in cancer cells, thereby inhibiting the expression of related proteins and reversing cell resistance. Next, we will describe the targets one by one.

2.1 ATP-binding cassette transporter

As the understanding of cancer biology has improved, different mechanisms have been proposed for the development of multidrug resistance in tumors. One of the traditional mechanisms is the overexpression of transmembrane ATP-binding cassette transporters, which use the energy released by ATPase hydrolysis to actively transport chemotherapeutic drugs across the cell membrane, reduce the drug concentration in tumor cells, reduce the accumulation of intracellular drugs, and lead to the failure of chemotherapeutic drug treatment^[4].

P-gp, also known as ABCB1/MDR1 protein, is a transmembrane glycoprotein with molecular weight of 170 kDa encoded by MDR1 gene. It is a very representative member of ABC transporter, which can transport a variety of hydrophobic substrates and anticancer drugs, including etoposide, doxorubicin and vincristine. Another member of the ABC transporter family, MRP1, also known as ABCC1, can transport glutathione conjugates and cyclic nucleotides, as well as a variety of other substrates, such as organic anions and a variety of chemotherapeutic drugs, such as doxorubicin, vincristine and colchicine^{[40⇓⇓~43]}. In 1998, Doyle et al. Isolated a new ATP-dependent transporter from drug-resistant breast cancer cell line MCF/AdrVp, which was named BCRP, also known as ABCG2 protein. Together with P-gp and MRP1, it constitutes the three major efflux transporters^{[44⇓⇓~47]}.

2.2 Proteins associated with apoptosis

Tumor cells will adopt a variety of strategies to resist apoptosis induced by chemotherapeutic drugs, in addition to the above-mentioned efflux pump mechanism, there are also activation of anti-apoptotic pathways. Apoptosis is the most common type of programmed cell death and an important component of the cell cycle. Many proteins are involved in the activation of apoptotic cascades. Cancer cells activate the anti-apoptotic pathway through the high expression of Bcl-2 family related proteins (such as Bcl-2, Mcl-1), Survivin and PLK1 proteins related to cell division, which lead to abnormal regulation of apoptosis and inhibit drug-induced apoptosis, resulting in drug resistance of cancer cells^{[48⇓⇓~51]}.

2.3 Other targets

Cytosolic Ca²⁺ is a key signal transduction regulator in various cancer processes including cell proliferation, tumorigenesis and migration. In cancer cells, many transmembrane Ca²⁺ channels or pumps responsible for Ca²⁺ transport are abnormally expressed, resulting in Ca²⁺ influx, promoting cancer cell proliferation and new survival pathways after drug action, thus leading to drug resistance^[52]. Among the different Ca²⁺ channels, transmembrane low-voltage activated T-type Ca²⁺ channels are highly expressed in a variety of cancer cells, which promote the proliferation of cancer cells by up-regulating the concentration of Ca²⁺ in the cytoplasm^[53]. Therefore, targeting related channel proteins to regulate cytosolic Ca²⁺ concentration may be a potential therapeutic target for reversing drug resistance. In addition, related work has targeted protein kinase B (AKt), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxia-inducible factor-1 (HIF-1), Xeroderma pigmentosum group F coupling factor recombinant protein (XPF), and Aurora kinase A (AURKA) to reverse chemoresistance^[54]^[55]^[56]^[57]. In recent years, with the deepening of the study on the mechanism of drug resistance, it has been found that some long noncoding RNAs are also closely related to drug resistance. For example, Liu et al pointed out that cancer susceptibility 11 long noncoding RNA mediates the resistance of hepatocellular carcinoma to carboplatin by up-regulating miRNA-21^[58].

3 Nanocarrier for co-loading siRNA and chemotherapeutic drug

Usually, chemotherapeutic drugs and siRNA can be loaded on different nanocarriers through physical or chemical interaction. Next, according to the types of nanocarriers used, we will introduce some of the latest research progress in the reversal of tumor drug resistance by co-loading chemotherapeutic drugs and siRNA.

3.1 Mesoporous silica nano-carrier

In recent years, mesoporous silica nanoparticles (MSN), due to its unique high porosity, biostability and non-toxic characteristics, are gradually being widely used in the cell delivery of siRNA and small molecule chemotherapeutic drugs^[59]. MSN can be divided into large pores and small pores in structure. MSN with small pore size can only encapsulate small molecular chemotherapeutic drugs, but can not encapsulate larger siRNA, but can adsorb siRNA on the modified surface through electrostatic interaction.

In recent years, a large number of studies have been reported on the co-loading of siRNA and chemotherapeutic drugs by modifying MSN surface. Meng et al. Wrapped MSNs with polyethylenimine-polyethylene glycol (PEI-PEG) to form a cationic surface, and then combined with negatively charged P-gp siRNA. On the other hand, DOX was adsorbed on the phosphate-coated pores through electrostatic interaction, so DOX was released only in the acidic microenvironment unique to tumors. They explored the co-loading of P-gp siRNA and DOX on MSN to overcome the resistance of breast cancer to DOX^[60]. In addition, they continued to use the same strategy to explore the effect on drug-resistant squamous cells, and in this work, the combination of different types of siRNA related to drug resistance with DOX was highlighted to determine the best drug combination, pointing out that siRNA loading P-gp, Bcl-2, c-Myc and PXR related to drug resistance.The combination of P-gp siRNA and DOX showed the best synergistic effect in vitro screening, which greatly improved the sensitivity of tumor cells to DOX. The system showed a rare effect of overcoming tumor heterogeneity in vivo, and had a good stealth effect, which reduced the uptake of drugs by RES system, so that the drug accumulation in tumor sites reached 8% of the injection dose.

Chu Xia et al. First loaded DOX in the gap, and then synthesized an ultra-thin MOFs material ZIF-8 membrane (zeolite imidazole skeleton-8) in situ on the MSN surface by optimizing the functional groups, reaction time and reaction temperature on the MSN surface.The negatively charged surface of the carboxylated MSN is reversed to a positively charged surface, so that the siRNA targeting Bcl-2 is electrostatically adsorbed thereon and can act as an encapsulating void (fig. 1)^[61]. After DOX-MSN-COOH @ ZIF-8/siRNA enters cells, the ultrathin ZIF-8 membrane can be decomposed in acidic lysosomes, releasing siRNA and chemotherapeutic drugs. In cytotoxicity assays, the DOX,IC₅₀ values of Dox-MSN-COOH @ ZIF-8/siRNA were only slightly decreased relative to Dox-MSN-COOH @ ZIF-8 and free for wild-type breast cancer cell line MCF-7,However, the IC₅₀ value was significantly decreased in the DOX-resistant breast cancer cell line MCF-7/ADR, and a similar phenomenon was also observed in the human ovarian drug-resistant cancer cell line SKOV3/ADR, which fully proves that the system has a significant effect on reversing drug resistance.

Li Linlin et al synthesized MSN nanocapsules (MSNC_S) by two-step alkali selective etching method. In order to improve the stability of nanoparticles, part of the residual amino groups of organosilicon were combined with PEG to form pMSNC_S, and then DOX was loaded into the pMSNC_S.Because a part of amino groups remain on the PEG-bound MSNCs and the PEG-bound MSNCs have a positively charged surface, negatively charged siRNA is further bound, and finally the siRNA targeting the T-type Ca²⁺ channel is loaded on the pMSNC/DOX through electrostatic interaction^[62]. Under different pH values, the hydrogen bonding and electrostatic interaction between DOX and silicon dioxide are changed, and when the pMSNC/DOX/siRNA is placed in a weakly acidic tumor cell microenvironment, DOX is released in the cell, so that the pH-sensitive DOX release is realized. In this work, the anti-tumor effect of pMSNC/DOX/siRNA was evaluated by CCK-8 test in vitro, and it was pointed out that the synergistic effect of siRNA targeting T-type Ca²⁺ channel and DOX could reverse DOX resistance only in drug-resistant cells.In non-MDR cells, the effect was only additive, which may be due to the G0/G1 arrest of MCF-7/MDR cell cycle caused by the intracellular release of siRNA, thus increasing the accumulation of intracellular drugs and synergistic killing effect. The in vivo anti-tumor experiment in mice further showed that pMSNC/DOX/siRNA had obvious anti-tumor effect, and the inhibition rate was as high as 76%.

In addition to the use of electrostatic interaction to achieve co-loading of siRNA, in order to further solve the problem that siRNA is easily degraded when exposed on the surface, large-sized mesoporous organosilica nanoparticles (MONs) are gradually used as co-loading carriers to encapsulate both siRNA and chemotherapeutic drugs^[63]^[64]. For example, Shi Jianlin et al. Developed a kind of macroporous hollow mesoporous organic silica nanoparticles (HMONs), which are macroporous but do not wrap siRNA in them^[65]. They modified disulfide bonds and poly (β-aminoester) (PAE) on the surface of silicone to form a new nanoparticle (HMONs-ss-PAE). PAE is a polycationic molecule composed of amino segments and degradable ester bonds, which can be used to bind siRNA, while DOX is encapsulated in the pores, so HMONs-ss-PAE can co-load DOX and P-gp siRNA. In this study, the encapsulated siRNA targeting P-gp down-regulated the expression of P-gp by 90%, while HMONs-ss-PAE significantly reduced the level of ATP, cut off the energy supply for P-gp drug efflux, and increased the uptake of DOX by drug-resistant cells in two ways. In exploring the effect of tumor inhibition, the work also showed that siRNA/DOX @ HMONs-ss-PAE had a strong killing effect on drug-resistant cells.

In terms of using MON as a carrier, in addition to co-loading, Shi Jianlin and others have further achieved the gradual release of siRNA and chemotherapeutic drugs, which is a relatively new development direction that must be considered in co-loading^[36]. They designed a spatially hierarchical core-shell MSN (core)/MON (shell) nanosystem (called H-MSNs), in which the core and shell of H-MSNs have mesopores of different sizes, and the core of MSN of H-MSNs is a small pore.The nano carrier is used for storing small molecule DOX, and the part of the MON which is wrapped outside and used as a shell is a relatively large pore; and polyethyleneimine (PEI) is grafted on the surface, so that the biocompatibility of the nano carrier drug is optimized, and the nano carrier drug can be combined with large-size siRNA targeting P-gp. The shell of MON has a complex disulfide hybrid skeleton, which will be broken in the high reductive microenvironment of tumor cells, so that siRNA in the shell is released first, down-regulating P-gp, and then DOX in the nucleus is gradually released. This unique design can reverse the drug resistance state through the action of siRNA, reduce drug efflux, increase the content of chemotherapeutic drugs in cells, and improve the sensitivity of cells to chemotherapeutic drugs.Thereby exerting the cytotoxic effect of chemotherapeutic drugs to kill cells, achieving the best effect of killing drug-resistant cells with the least chemotherapeutic drugs, and avoiding stimulating cells to produce stronger drug resistance again as far as possible.

In addition to MSN, other similar inorganic nanomaterials have also been used for co-loading of siRNA and chemotherapeutic drugs. For example, Chowdhury et al. achieved co-loading of PTX and siRNA using pH-dependent carbonate apatite (CA) nanoparticles^[66]. Selenium is an essential trace element for humans and animals, and clinical trials have proved that selenium compounds can be used for cancer treatment. Liu Jie and others used layered double hydroxide nanoparticles (LDHs) to synergistically deliver selenium (Se) and siRNA. They first embedded Se into the layers of LDHs to form Se @ LDH, and then complexed siRNA through electrostatic adsorption.It is pointed out that Se @ LDH may be a microtubule stabilizer, which inhibits cell proliferation by blocking G2/M cell cycle, disrupts the formation of spindle in normal mitosis, and induces cell apoptosis, while siRNA is easily adsorbed on Se @ LDH, and siRNA reverses tumor drug resistance on the other hand^[67]^[68]. Zhang et al. Reported a Ca/P nanosystem, which was prepared by first using Ca²⁺ to form a complex with siRNA, then reacting with phosphate ions to form a Ca/P/siRNA core, coating the outer layer with lipids for stability and loading DTX in the lipid layer, which was characterized by the fact that the lipid layer was composed of DOPA/RGD-PEG-DSPE.The RGD (arginine-glycine-aspartic acid) can target the tumor, increase the enrichment of the nano-drug in the tumor site in the blood circulation, improve the drug uptake of tumor cells, and the apoptosis rate is as high as 66%, which can well inhibit the growth of tumor tissues in vivo^[69].

3.2 Metal organic framework

metal-organic frameworks (MOFs) have the characteristics of self-assembly, porous and easy to be modified. In recent years, they have been gradually developed to nanoscale, resulting in nanoscale metal organic frameworks (nanoMOFs). These nanosystems generally have the characteristics of diverse structures, excellent loading capacity and good biodegradability, and have been used as carriers for delivering chemotherapeutic drugs^[70,71]. The UiO series in the MOF material is formed by using Zr₆(μ₃-O)₄(μ₃-OH)₄ as a secondary building unit (SBU) and dicarboxylate as a bridging ligand, and has a hexagonal plate shape. Lin Wenbin et al. Used encapsulation to load cisplatin prodrug on UiO, and combined siRNA targeting multiple drug resistance genes (P-gp, Bcl-2, Survivin) to MOF through the formation of multiple coordination bonds between the phosphate residues on the siRNA backbone and Zr on the surface of MOF, thus constructing nanoMOFs drugs to reverse drug resistance in ovarian cancer^[72]. There are some differences between this work and many previous reports, the most prominent of which is that they load a variety of siRNAs (including Survivin, Bcl-2 and P-gp siRNAs) related to drug resistance reversal on the same vector at the same time.Moreover, the siRNA loaded on the UiO NMOFs material can easily escape from the cell endosomes or lysosomes. In order to further explore the application of the UiO NMOFs material in this field, we can next compare the difference in the effectiveness of reversing drug resistance between loading an equal amount of siRNA and loading multiple siRNAs. Among MOF materials, MIL-101 is also a promising nano-carrier material. The narrow mesoporous cavity can be used to load small molecular drugs, and the metal ions on the external surface can bind biological macromolecules such as siRNA. Liu Jie et al synthesized a selenium-ruthenium nanoparticle modified MIL-101 (Fe), which used the anti-tumor effect of selenium and ruthenium, and delivered siRNA targeting P-gp to reverse drug resistance^[73]. Huo Jia et al. Developed a multifunctional metal-organic framework (AMOFS) responsive to azoreductase, which can encapsulate DOX, and can bind siRNA and CPPs targeting HIF-1α on its surface through electrostatic and coordination interactions^[74]. (DRHC in fig. 2 refers to a DNA/RNA hybrid complex, herein referred to as siRNA). AMOFS is coordinated by Fe (Ⅲ) and azobenzene-4,4 '-dicarboxylic acid. In the hypoxic tumor microenvironment, azoreductase is highly expressed, azobenzene is degraded under the action of the azoreductase, DOX and siRNA are promoted to be released, and the MOF-based hypoxic response nano-drug is constructed. In the case of rapid growth of solid tumors, hypoxia is their typical physiological characteristics, and the nano-probe is designed according to the physiological characteristics of solid tumors, has high specificity, and combines CPPs as target molecules on the probe.Nanoparticles can directly enter the cytoplasm without consuming energy, deliver chemotherapeutic drugs into cells, overcome drug resistance through Trojan horse drug delivery, and have the dual characteristics of targeting and specific response. HIF-1α can enhance the expression of P-gp in tumor cells, and siRNA targeting HIF-1α can not only silence the expression of HIF-1 α, but also down-regulate the expression of P-gp, thus reversing the drug resistance of tumors at the gene level. The survival rate of MCF-7 cells treated with the nano-drug was only 29%. In vivo experiments, the survival rate of mice in the nano-drug group reached 100% one month after the death of mice with tumors in the control group, achieving a relatively ideal therapeutic effect.

3.3 Polymer micelle

Polymer micelles are composed of amphiphilic polymers, which can self-assemble to form core-shell nanospheres in aqueous environment. In general, the copolymers contain cationic hydrophilic moieties that complex with the siRNA, and hydrophobic moieties that drive self-assembly and encapsulation of the drug. At present, micelles formed by diblock copolymers or triblock copolymers are the most widely studied co-loading carriers of siRNA and chemotherapeutic drugs. Cationic polymer micelles are the most common carriers for co-loading siRNA and chemotherapeutic drugs, but they are difficult to release in cells due to their strong adsorption on siRNA and chemotherapeutic drugs, thus limiting their direct application in this field. Therefore, as the study of responsive micelles is more and more extensive, more and more attention has been paid to the response conditions of drug release, among which the most widely used is GSH with gradient differential expression in and out of tumor cells.As well as the acidic environment in cancer cells, we will elaborate on the application of responsive micelles in drug delivery through the work of Zhang Xuenong and Zhou Jianping.

Zhang Xuenong et al synthesized a binary polymer composed of low density lipoprotein (LDL) and N-succinylchitosan-cystamine-uric acid (NSC-SS-UA)^[75]. NSC-SS-UA itself can be used as a triblock micelle unit, NSC has hydrophilicity, and SS-UA acts as a hydrophobic core to drive self-assembly and load PTX. The disulfide bond of SS in SS-UA can be broken by intracellular GSH, showing redox sensitivity, while UA shows pH sensitivity, so NSC-SS-UA becomes a triblock micelle unit loaded with PTX with redox and pH sensitivity. However, because NSC-SS-UA could not load BCRP siRNA, they first bound siRNA to cholesterol (CHOL) to construct a lipophilic siRNA, and then loaded it into the lipophilic interior of LDL to form a siRNA-loaded LDL unit. Finally, the LDL moiety was combined with the NSC-SS-UA moiety through amide bonds to form a binary polymer containing micelles. The binary polymeric micelles have many advantages over other conventional nanocarriers. For example, LDL in the system not only acts as a carrier of siRNA, but also targets LDL receptors highly expressed on the surface of tumor cell membranes, which improves the uptake rate of drugs by cells compared with ordinary nano-carrier drugs. Moreover, as a substance extracted from serum, LDL has no toxicity and specificity, reduces the uptake of RES system, increases the circulation time in vivo, and improves the retention in tumor sites. In addition, acidity is a characteristic physiological parameter of tumor microenvironment. LDL-NSC-SS-UA is sensitive to both pH and redox. When it reaches the cells, the drug is released in the acidic environment with high GSH and tumor cells. In vitro validation experiments showed that the micelles without any drug loading had good biological safety, while the cell survival rate of siRNA-PTX/LDL-NSC-SS-UA group was only 30. 47% and 15. 59% after incubation for 12 H and 24 H, which was consistent with the anti-tumor results in vivo, indicating that siRNA could maintain a higher intracellular drug concentration, enhance cytotoxicity and reverse the drug resistance of breast cancer drug resistant cells MCF-7/Taxol after reducing the expression of BCRP.

In addition to the targeting of LDL as a carrier mentioned above, some researchers will modify specific substances with targeting tumor cells on the surface in order to further enhance the targeting effect of micellar carrier drugs, such as the following work of Feng Sishen and Zhou Jianping. Feng Sishen et al. Developed a micellar system that combines the advantages of targeting and co-loading (Fig. 3)^[76]. First, the siRNA targeting PLK1 was combined with vitamin E (TPGS) through disulfide bonds to form a diblock polymer (siPLK1-ss-TPGS), which was then mixed with anti-cancer drug DTX to form a new micelle, and then Herceptin (antibody drug) was combined with the co-loaded micelle synthesized in the previous step to endow it with targeting to HER2-overexpressing cancer cells. By adjusting the ratio of DTX and siPLK1-SS-TPGS: TPGS, the loading of DTX and siRNA can be precisely controlled. In addition, the co-administered micelle formulation (TPGS-DTX-siPLK1, micSD) was shown to be more effective than free DTX and the commercial DTX-loaded micelle formulation (TPGS-DTX, micD) in terms of cytotoxicity, with the IC₅₀ of DTX loaded in micSD being 537-fold lower than that of DTX alone after co-incubation with breast cancer cells SK-BR-3 for 24 H. Zhou Jianping et al. synthesized an amphiphilic conjugate based on hyaluronic acid (HA), HA-ss- (OA-g-bPEI) (HSOP), which can realize the co-delivery of PTX and siRNA targeting AURKA^[77]. The polymer micelle is characterized in that the polymer micelle has targeting property and redox sensitivity, and the disulfide bond in the micelle is broken to release the medicament by utilizing the characteristics of gradient difference of GSH content inside and outside cells and higher reduction potential inside the cells; In addition, the micelle has another advantage that the HA shell with hydrophilicity has targeting property to receptors such as CD44, RHAMM and the like which are highly expressed on tumor cell membranes.

Zhang Xin et al. coupled Dox to stearoyl chloride (C18) by crosslinking agent N-methyldiethanolamine (N) to form C18-N-Dox, which can self-assemble into micelles, but in order to maintain stability, it was mixed with DSPE-PEG2000 to prepare micelles^[78]. The tertiary amine of C18-N-DOX is capable of complexing siRNA targeting PLK1 depending on the positive charge provided under acidic conditions (pH = 3), thus achieving co-loading of siRNA and Dox. In vivo experiments showed that the polymer micelle drug could completely inhibit tumor growth in HeLa female BALB/C nude mice within 18 days. Li et al. synthesized a pH-sensitive amphiphilic polymer, in which the hydrophobic block contains repeating units of DPA groups, and the hydrophilic block is low molecular weight PEI, which forms micelles through self-assembly, loads PTX at neutral pH, and then aggregates siRNA targeting Akt in the micelles through the cationic charge of PEI^[79]. Sun et al. synthesized a triblock copolymer micelle :mPEG₄₅-b-PCL₈₀-b-PPEEA [poly (ethylene glycol) -b-poly (ε-caprolactone) -b-poly (2-aminoethylvinyl phosphate)] with a core-shell structure, which can co-load siRNA and PTX targeting PLK1 (fig. 4)^[80]. After 32 days of injection, the tumor growth was completely inhibited by the micelles loaded with both drugs, but the xenograft tumor treated with the micelles loaded with a single drug grew to about three times the original volume.

3.4 Liposome/vesicle

Since the FDA approved the use of Doxil^® (doxorubicin liposome injection) for cancer treatment, the study of liposomes as gene and chemotherapeutic drug carriers has become more extensive^[81]. Liposomes have a hydrophilic core region, which can be loaded with hydrophilic drugs such as siRNA, DOX, etc., while the hydrophobic region between the bilayers can be used to load lipophilic drugs such as PTX, and siRNA can also be loaded by reasonably modifying the surface of liposomes. The greatest advantage of liposomes is their biocompatibility, which can bypass the "clearance guards" in vivo, increase the circulation time in vivo and the amount of retention in the tumor site. The applied liposomes include cationic liposomes and solid liposomes. Cationic liposomes have a high affinity for the slightly negatively charged cell membrane and can enter the cell by endocytosis or fusion and escape from the endosome with high efficiency.

Liposomes have been widely used as co-loading carriers. When liposomes are formed, the inner layer similar to phospholipid bilayer can be formed first, and hydrophobic drugs can be loaded in the core. Then the outer layer of bilayer can be constructed by self-assembly of hydrophobic interaction between lipids. At the same time, lipophilic drugs can be loaded in the hydrophobic region in the middle of bilayer. He Zhonggui et al. First used DOTAP, DPPC and CHOL to form the inner layer of the bilayer, and wrapped siRNA in the hydrophilic core, then used DSPE-mPEG and DPPC to form the outer layer of the bilayer through hydrophobic self-assembly, and wrapped lipophilic PTX between the two hydrophobic layers to form an asymmetric membrane of siRNA liposomes encapsulating low-dose PTX and targeting GAPDH^[82]. Zhu Guangyu et al. used DOTAP to encapsulate siRNA and cisplatin succinate (a cisplatin prodrug) in the hydrophilic core to form the inner part of the bilayer, and then on the basis of the previous one,DOPE and DSPE-mPEG use hydrophobic interaction to form an outer part equivalent to a phospholipid bilayer, in which the modification of PEG improves biocompatibility and in vivo circulation time, and finally CHOL is inserted in the hydrophobic region between the two layers to stabilize the structure (Fig. 5)^[83]. Under the 300 nmol·L^-1siRNA concentration, the survival rate of A549 cells was only 20%, and the IC₅₀ value was 85 times higher than that of the small molecule prodrug. Cui Jingnan et al. prepared a cationic liposome. In the preparation of co-loading system, protamine (PRTM) was first selected to concentrate siRNA into a compact "core", and then cationic lipid linked with carbamate (DDCTMA) was used to wrap the compact "core", and lipophilic PTX was loaded between the hydrophobic layers in the lipid bilayer^[84]. Wang et al. Used DOTAP, CHOL and PRTM to make cationic liposome nanoparticles. First, PRTM bound siRNA under electrostatic action, and then encapsulated Gem in the hydrophobic part of the phospholipid bilayer of liposomes to form LP-Gem-siMcl-1^[85]. When LP-Gem-siMcl-1 was incubated with pancreatic cancer cells PANC-1 and BxPC-3 for 72 H, the inhibition rates were 78% and 79%, respectively. In vivo experiments further showed that the combination therapy based on LP-Gem-siMcl-1 showed better synergistic anti-tumor effect than LP-siMcl-1 or LP-Gem alone.

Most liposomes, as carriers, will wrap siRNA in the core as a hydrophilic substance. Different lipids are used, and some will adsorb siRNA on the surface of liposomes. For example, Hao Jihui et al. Used biocompatible lipid-polymer hybrid nanoparticles to co-deliver siRNA and Gem targeting HIF-1 α^[86]. They first used mPEG-PLGA (lactic acid-glycolic acid) to construct liposomes, with a hydrophilic core in the center, loaded with Gem, combined with epsilon-polylysine (EPL) in the hydrophobic outer layer, EPL is cationic, used to adsorb negatively charged siRNA, forming ENP, and finally wrapped with PEG-modified lipid bilayer in the outermost layer to form LENP, and studied its therapeutic effect in subcutaneous and in situ tumor models. The lipid bilayer can protect the siRNA from degradation, and other negatively charged substances in the serum can replace the siRNA, prevent the leakage of drugs, and even provide a "stealth" function from the binding of proteins in the serum. The most desirable aspect of this work is that any material they use is FDA-approved and has high safety and biocompatibility. Zhang can et al. Used redox sensitive cationic oligopeptide liposomes (LHSSG2C14) and natural soybean phosphatidylcholine (SPC) to form a new liposome-based nanosystem for co-loading PTX and Survivin-targeted siRNA, inserting PTX between hydrophobic layers and coating siRNA on both the core and the positively charged surface^[87].

Like the above micelles, MSN and other nanocarriers, when combined with targeted molecules, the nanocarrier drugs will not only stay in the tumor tissue through the EPR effect, but also promote the nanocarrier drugs to have a higher cell uptake rate. Therefore, researchers have also considered combining target molecules with liposome carriers to improve their cellular uptake. Gao et al. First formed a DOX-containing multilayer assembly liposome, then extruded the monolayer liposome, loaded PRTM and hydrophilic siRNA into the hydrophobic core, and finally fused the liposome that had bound the target molecule Fab ' (targeting the high expression of epidermal growth factor (EGFR) in the liver cancer cell membrane) with the liposome that had loaded siRNA and DOX.The targeted liposome complex co-loaded with RRM2 siRNA and DOX was formed, and its therapeutic effect was greatly enhanced compared with the non-targeted or single-drug control group^[88].

The solid lipid nanocarrier in liposomes is composed of lipids that remain solid at body temperature, which is suitable for hydrophobic drug delivery, and then modified with cationic surfactants to form a positively charged surface, which can be used to deliver nucleic acids^[89]. Yu et al. achieved co-loading of PTX and Mcl-1 siRNA with cationic solid lipid nanoparticles, in which cationic lipid 1,2-dioleoyl-sn-glycero-3-ethylphosphine choline (EDOPC) was used as a surfactant to complex siRNA, and then PTX was encapsulated in a hydrophobic core by solvent emulsification^[90]. Nam et al. Synthesized LDL-like SLNs with stable core-shell structure by recombining LDL. The core can be used to encapsulate hydrophobic anti-cancer drug PTX and optically active nanocrystalline CdSe/ZnS quantum dots. The cationic surface can be compounded with multi-ion macromolecules such as siRNA and DNA through electrostatic interaction to obtain a nano-composite system (PQSLN/Bcl-2 siRNA) loaded with siRNA targeting Bcl-2, PTX and quantum dots^[91]. After the nanocomposite system enters the cancer cells, the expression of Bcl-2 protein is obviously down-regulated, the sensitivity of human lung cancer cells A549 to PTX is greatly improved, and the inhibition rate of the nanocomposite system on the A549 cells is as high as 70%, while the inhibition rate of a nanosystem (PQSLN) which is not simultaneously loaded with siRNA on the A549 cells is only 30%. The IC₅₀ of PQSLN/Bcl-2 siRNA on A549 cells was 225 ng·mL^-1, which was about 20-fold lower than the IC₅₀ of PTX preparation (4.6 μg/mL). In addition, the fluorescence signal of quantum dots can also be used to realize the function of in situ imaging.

The structure of vesicles is similar to that of liposomes, but they are more stable than liposomes, and compared with micelles, vesicles can encapsulate both hydrophobic and hydrophilic substances. Vesicles can also effectively increase drug retention in the body and reduce drug clearance in the body. However, because its surface is neutral, it can not adsorb siRNA through electrostatic interaction. To solve this problem, researchers usually modify its surface to make it have the function of gene delivery.

Sun et al. designed a nonionic surfactant vesicle co-loading system, which reversed the sensitivity of cancer stem cells 231-CSCs to DOX by loading two kinds of siRNA to down-regulate the expression of drug resistance genes BCRP and Bcl-2^[92]. In this co-loading system, they incorporated DOTAP-cationic lipids into Span80 vesicles that could not be directly used for gene delivery, allowing DOX encapsulation and siRNA complexation to occur simultaneously. In the co-loading system, siRNA targeting BCRP mRNA serves as an efflux pump inhibitor, and siRNA targeting Bcl-2 mRNA serves as an inhibitor of anti-apoptosis defense, so that the apoptosis effect of DOX is synergized, and a better anti-cancer effect is achieved. Ordinary vesicles are generally neutral and can not be directly used as co-loaded nanocarriers, but polymer vesicles are recognized as good nanocarriers for synergistic delivery. Kim et al. designed polymer vesicles, and they encapsulated siRNA targeting Bcl-xL and DOX into vesicles composed of methoxy polyethylene glycol-b-poly D, L-lactic acid (mPEG-b-PLA) to achieve co-loading (Fig. 6), which had a good inhibitory effect on the proliferation of gastric cancer cells, showing the potential of polymer vesicles as nanocarriers^[93].

3.5 Dendrimer

Dendrimers are nanoscale macromolecules with dendritic structure, narrow size and polydispersity, and the size of dendrimers can be controlled by changing the number of layers of the tree to meet different application needs.Its terminal structure can be modified to load nucleic acids, proteins and other drugs, and the internal voids can also wrap drugs through interaction. poly (amido amine) (PAMAM) is a promising co-loading nanocarrier, in which hydrophobic drugs with small molecular weight mainly interact with PAMAM molecules, and negatively charged nucleic acids interact with cavities composed of a large number of primary amine groups on the surface^[94]. Based on this design concept, some foreign researchers have synthesized a triblock fourth-generation PAMAM dendrimer, which can be used for siRNA delivery, and further designed a mixed micelle system to achieve the co-loading of chemotherapeutic drugs and siRNA^[95].

Mohammed et al. Have also done some work on anti-cancer research by using dendrimers as carriers to load drugs. One of their first reported work was the synthesis of a folate-polyethylene glycol-glucose dendrimer conjugate with glucuronosyl-β-cyclodextrin (Fol-PEG-GUG-β-CDE) (Generation 3), which can be used as a vector for siRNA targeting PLK1. In the drug delivery system, the GUG-β-CyD moiety can interact with the endosome membrane, resulting in its destruction, making it easier for the siRNA-loaded complex to escape from the endosome and play its role in gene regulation. In addition, the combination of folic acid in the system increases the uptake of the drug-loaded system in cancer cells and enhances the RNA interference effect of siRNA. In their latest work, they used the previously synthesized vector to achieve the co-loading of DOX and siPLK1^[96]. Studies show that the Fol-PEG-GUG-beta-CDE/siPLK1/DOX ternary complex shows good anti-cancer effect in vitro cell experiments, the inhibition effect on human oral epidermoid carcinoma cell KB is as high as 94%, and experiments on tumor-bearing mice further prove that the ternary complex obviously inhibits the growth of tumors and shows strong anti-tumor activity in vivo.

3.6 Layer-by-layer assembly of nanoparticles

layer-by-layer (LbL) nanoparticles are a novel drug delivery platform, which use the process of sequential deposition of oppositely charged polymers to construct high RNA content and high stability films on the nanoparticle core. The modular design of the LbL nanoparticle platform enables the introduction of therapeutic drugs in both the core and surrounding layers, creating a tunable multi-drug delivery system, such as the construction of RNA-containing layer-by-layer assembly films on a core loaded with chemotherapeutic drugs (Fig. 7)^[97]. In line with the above ideas, Venuganti et al. Constructed a layer-by-layer assembly of gold nanoparticles (LbL-AuNP) containing siRNA targeting STAT3 and imatinib mesylate (IM) for the treatment of melanoma resistance^[98]. Hammond et al. prepared layer-by-layer nanoparticles of DOX and siRNA targeting MRP1 by coating poly L-arginine (PLA)/siRNA/PLA/HA membrane on DOX liposomes^[97]. In the synthesized LbL liposome preparation, the loading percentage of DOX is up to 5.5% w/w, and each PLA/siRNA bilayer contains 3500 siRNA molecules, so that the LbL liposome preparation has higher tumor inhibition efficiency, the release of siRNA is faster than that of DOX within 72 H, and the siRNA can obviously reduce the MRP1 mRNA level in tumors, so that DOX shows stronger cell inhibition rate. The experiment showed that the combination of MRP1siRNA and DOX not only increased the killing rate of Dox in vitro by 4 times, but also reduced the average tumor volume in vivo by 8 times compared with the control group.

3.7 Hyaluronic acid nanosystem

Hyaluronic acid (HA) is a natural, high molecular weight, anionic biopolymer found in the extracellular matrix of the human body. HA polymers have abundant binding sites, such as -COOH and -OH, and can be synthesized into a variety of artificially designed derivatives. Another prominent advantage of HA is that it can specifically bind to CD44 receptor, which is highly expressed on the surface of cancer cells, and enter cells through endocytosis. In addition, HA as a nano-carrier also has the advantages of biodegradability, non-immunogenicity and non-toxicity.

In general, the anionic properties of HA can be used to assemble positively charged nanocarriers with positively charged substances through electrostatic interaction, which can be used for cell delivery of siRNA. For example, Yang et al. designed HA-PEI/HA-PEG derivatives, which can be used to deliver siRNA targeting P-gp^[99]. Chen et al. designed a nano-delivery system of CaP-coated DPA/Zn-labeled HA-NP (CaP-HDZ) for siRNA targeting P-gp and DOX co-transport^[100]. The nano delivery system is mainly composed of three parts: firstly, hyaluronic acid-5 beta cholic acid conjugate (HA-CA) is self-assembled to obtain hyaluronic acid nanoparticles (HA-NPs) with tumor targeting, and hydrophobic small molecular drugs can be wrapped in the core of the HA-NPs; Then Zn (II) -dipyridyl molybdic acid (DPA/Zn) capable of binding with siRNA is assembled; The outermost layer is coated with a CaP coating to effectively avoid the damage of the physiological environment to the nano system. The siRNA targeting P-gp in the CaP-HDz/Drug/RNA-NFs nano system constructed by the method regulates and controls the P-gp mRNA, so that the sensitivity of OVCAR-8/ADR cells to DOX is restored or even enhanced, thereby showing good anti-cancer activity in vitro and in vivo.

Wu Jingliang et al. synthesized a liver-targeting nanoparticle (GH-DPP) capable of simultaneously loading DOX and siRNA targeting Bcl-2. The nanoparticle comprises 1,2-diformyl-glycerol-3-phosphoethanolamine-polyethylene glycol-polyetherimide (DSPE-PEG-PEI) and glycyrrhetinic acid modified hyaluronic acid (GA-HA)^[101]. DOX was first loaded into the core of the DPP, then the DPP was loaded with siRNA via electrostatic interaction, and finally the GH-HA conjugate was encapsulated on the DPP nanoparticles (fig. 8). In vitro cytotoxicity test showed that siRNA/DOX/GH-DPP nanoparticles had better inhibitory effect on HepG2 cells than DOX or Bcl-2 siRNA alone when Bcl-2 expression was down-regulated by RNA interference due to the synergistic anti-tumor effect of DOX and Bcl-2 siRNA. In addition, compared with the siRNA/DOX/DPP nanoparticles without GA-HA coating, the siRNA/DOX/GH-DPP nanoparticles showed more cytotoxic activity on HepG2 cells with high expression of GA receptor due to their targeting.

3.8 Brief summary

In order to more clearly illustrate the differences between various co-loaded nanomedicines, the mechanism of drug release in cells, and the advantages and disadvantages of various nanocarriers, they are summarized in Table 1. In addition, the abbreviations appearing in the article are compiled in Table 2.

4 Extend

In the aspect of reversing tumor multidrug resistance, there are more and more studies on the synergistic effect of multiple therapeutic strategies by co-loading nanocarriers. In the aspect of co-loading of siRNA and chemotherapeutic drugs, in addition to using siRNA to interfere with mRNA expression to achieve the regulation of some classical sites, some researchers also use siRNA-based RNA interference technology to target some non-mRNA targets, such as microRNA. Ju Yixian et al. Synthesized multifunctional nanocomposites (PPG) with PEI, poly (4-styrenesulfonate) (PSS) and graphene oxide (GO), and then adsorbed DOX on PPG by physical mixing, and then immobilized siRNA targeting microRNA-21 on PPG by electroadsorption, thus increasing the accumulation of DOX in drug-resistant cells^[102]. In addition, in addition to loading siRNA to down-regulate the relevant mRNA to reverse the sensitivity of cells to chemotherapeutic drugs, some researchers use microRNA to regulate the nature of the corresponding mRNA, and consider co-delivery of microRNA and chemotherapeutic drugs in cells to study their synergistic anti-cancer effects. For example, Kim et al. Prepared a nanosystem co-loaded with microRNA and chemotherapeutic drugs^[103]. The system takes mesoporous titania supported on silicon as a core (MTNst), and then uses poly-L-lysine (PLL) and polyethylene glycol-b-poly-L-aspartic acid (PEG-b-PLD) to construct a layer-by-layer structure similar to a halo through the action of opposite charges. PTX is loaded in the MTNst, and the microRNA is adsorbed on the PLL layer through the charge effect, so that the synergistic effect of chemotherapy and gene therapy is realized, and good treatment potential is shown for colorectal cancer. Similarly, Zhao et al. 'S study attempted to reverse drug resistance by up-regulating the content of microRNA as a tumor suppressor gene in cells^[104]. They used fat-coated calcium carbonate nanoparticles loaded with the chemotherapeutic drug 5-fluorouracil and microRNA-375-3p to reverse resistance. The research on the use of nanocarriers to integrate different treatment strategies is not limited to the combination of gene therapy and chemotherapy. With the deepening of research, some researchers have even unified the three strategies of gene therapy, phototherapy and chemotherapy into one nanocarrier in reversing drug resistance of tumors, showing excellent anti-tumor effect^[105].

5 Conclusion and prospect

In the research of anticancer drugs, the application of nano-carrier platform has a significant role in promoting the targeted delivery of anticancer drugs. It not only helps to improve the stability of drugs, protect drugs from degradation and reduce adverse reactions, but also increases the accumulation of drugs in tumor sites through EPR effect. But more importantly, it can realize the co-delivery of siRNA and chemotherapeutic drugs, which can integrate different therapeutic mechanisms, overcome drug resistance, and realize Trojan horse drug delivery, which is of great significance in reversing drug resistance of tumors.

The co-loading of nanocarriers has been widely studied, but there are still many problems to be solved in order to achieve better therapeutic effect. (1) To find better targets for gene regulation, such as glutathione peroxidase (GPX4), which has recently been reported to be related to iron death^[106,107]. (2) For different types of tumors, what kind of chemotherapeutic drugs and what kind of siRNA can achieve the best therapeutic effect in the construction of the composite system, and what is the optimal loading amount of each drug, and whether the loading amount and compatibility should be considered when the therapeutic effect is not achieved. (3) In addition, stepwise release is a very important development direction in co-loading, and siRNA first improves drug resistance in many ways, such as reducing the expression of efflux proteins,The interruption of the anti-apoptotic pathway can maximize the cytotoxic effect of the limited chemotherapeutic drugs released next, and the smaller the dose of chemotherapeutic drugs, the more beneficial it is for cells to be in a sensitive state. (4) More problems should be considered in preparation research, such as whether siRNA is not stable enough only by electrostatic adsorption on the surface of the carrier, and the selection of materials with targeting properties such as HA to construct a protective layer or the selection of more stable binding methods, such as group modification of RNA and chemical bonding to the carrier; In order to realize the stepwise release of siRNA and chemotherapeutic drugs, it is necessary to load siRNA and chemotherapeutic drugs on the carrier and then freeze dry the whole complex system, but whether the stability of the complex system can be guaranteed in this process and whether leakage will occur; In addition, the uniformity of the prepared drugs should be ensured, the preparation steps should be simplified as far as possible, and the production cost should be reduced. To sum up, targeting, sustained release, stepwise release and optimal drug content and compatibility are the development direction of co-loading nano-carrier drugs.

As mentioned above, the generation of drug resistance is not only related to the changes in the content of relevant components at the cellular level, but also to the complex microenvironment of tumor tissues, which can affect the effect of drugs, and eliminating this effect is a very important link in the clinical application of nanocarrier drugs. Therefore, it is necessary and valuable to explore how to make the co-loading nanosystem play a better therapeutic role in the microenvironment of hypoxia, acidity, rigid extracellular matrix and vascular abnormalities, and further modify or optimize the co-loading nanosystem in the future. In terms of the construction of nano-systems, materials and chemistry researchers should also do more in-depth research on improving the stability, repeatability, biological safety and metabolism of nano-carriers after delivery.To develop a nano-carrier drug that can circulate in the blood, not be recognized by the immune system, and can identify the target of tumor tissue for effective drug delivery or gene silencing, so as to further promote its real clinical research process. In a word, the synergistic effect of gene interference and chemotherapy based on nanocarriers provides a new method and means to solve the drug resistance of tumors and completely cure cancer. With the deepening of research, we hope to make greater breakthroughs in practical applications.

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