Sheng-Nan Huang,Xia-Li Zhu,Hong Zhu,Jin Zhang, ,Jin-Xin Miao,Ming-San Miao

1Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450058,China;2College of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450058,China.

Various effective ingredients extracted from traditional Chinese medicine have achieved more and more attention in the field of cancer therapy due to their high bioactivity and good safety.These traditional Chinese medicine effective ingredients include paclitaxel,arsenical trioxide,camptothecin and so on.However,poor water solubility and low bioavailability of these traditional Chinese medicine ingredients severely hindered their clinical application.The rapid development of nanotechnology provided a new platform,new opportunities and new challenges for their effective delivery in vitro and in vivo.This minireview provides a concentrated summary of the most recent progresses made in this emerging field,highlighting the “combination ancient traditional Chinese medicine ingredients and advanced nanotechnology” strategy for favoring the construction of next generation cancer nanomedicines.

Key words:TCM effective ingredient,Nanotechnology,Paclitaxel,Arsenical trioxide,Camptothecin

Background

Recent campaign with COVID-19 has been witnessing the special potency of traditional Chinese medicine(TCM).This situation has also aroused a new wave of enthusiasm of scientists to explore ancient TCM that have already been approved as potential drugs.For thousands of years,under the guidance of the theory of Chinese medicine,TCM has played an important role in fighting diseases and protecting health in practice [1].TCM has played an important role in extending the survival period of cancer patients and improving their quality of life [2].With the continuous in-depth research on the chemistry and pharmacology of TCM,more and more effective ingredients of TCM have been discovered and confirmed.Drugs such as paclitaxel(PTX),arsenical trioxide (ATO) and camptothecin(CPT) derivatives have also been used for cancer therapy clinically [3-5].It is widely recognized in the world that ginsenoside,curcumin,quercetin,resveratrol,matrine,icariin and so on have also been studied as potential anti-tumor drugs [6].The anti-tumor activity and mechanism of TCM effective ingredients have the characteristics of multi-pathway,multi-target,and multi-pathway.TCM effective ingredients achieve anti-tumor effects through inhibiting tumor cell proliferation,inducing tumor cell apoptosis,inhibiting tumor cell invasion and metastasis,affecting autophagy,regulating reactive oxygen levels,anti-tumor angiogenesis,reversing drug resistance,regulating tumor microenvironment (TME),remodeling immunity and intestinal flora and so on [7,8].

Although many TCM ingredients have strong pharmacological effects in vitro,many problems such as poor water solubility,short half-life,poor stability,low bioavailability,and large toxic and side effects,severely hamper their drug ability and clinical application.Therefore,achieving effective delivery of TCM ingredients in vivo has become a key problem and an urgent problem for their clinical application.Nanotechnology is considered to be one of the three core technologies supporting human civilization in the 21st century,and has broad application prospects in the field of biomedicine,such as drug delivery,bioimaging,disease diagnosis and precision treatment [9].TCM ingredients-based nanomedicine mainly includes:(1)nanometerization of the drug itself,in other words,the drug is made into nanometer size by a certain method to increase the specific surface area and improve absorption;(2) apply nanocarrier to load drugs,such as liposome,nano-emulsion,nano-micelle,inorganic nanoparticles,exosomes and so on;(3) conjugate drugs with polymers or small molecules,or cross-link with different drugs to form nanoparticles self-assembly via noncovalent interactions.Nanomedicines can improve anti-tumor efficacy and reduce side effects through their unique advantages,such as improved solubility and stability,enhanced ability to penetrate various barriers,extended circulation time in vivo,enhanced drug accumulation in tumor site,stimuli-responsive drug release behavior,combination therapy with different treatments and so on [10,11].

In this minireview,we will provide a concise but concentrated summary of nanomedicines engineered with TCM effective ingredients (e.g.,PTX,ATO and CPT) developed in the recent years for enhanced cancer therapy.The most recent advances in the fabrication of TCM ingredients-based cancer nanomedicines will be presented (Figure 1).It is expected that the clinical translation of nanomedicines can be accelerated by combination of TCM and nanotechnology with elevated cancer therapeutic efficacy and reduced side effects.

Therapeutic applications of TCM effective ingredients-based cancer nanomedicine

PTX

Figure 1 Overview of new cancer nanomedicines by combination ancient TCM effective ingredients and advanced nanotechnology.TCM,traditional Chinese medicine.

PTX is one of the most widely used chemotherapeutic agents and is the first or second line clinical treatment for several types of cancers.PTX has been formulated and marketed as Taxol® at first and then prepared as nano-formulations,which are approved for clinical use or are undergoing clinical development.These PTX nano-formulations include albumin-based nanoparticle(Abraxane),micelle (Cynviloq,Paclical,NK105 and Triolimus),liposome (Lipusu,SB05 and LEP-ETU),polymeric-lipidic nanoparticle,emulsion (DHP-107)and so on [12].Despite the significant number of PTX nano-formulations have achieved success,researchers’enthusiasm for improving PTX delivery and efficacy through nanotechnology remains high [13,14].In addition to proteins,liposomes,and polymers,researchers have also attempted to apply RNA,DNA,and peptides as nanocarriers to improve PTX delivery and cancer treatment [15-20].In order to overcome the complexity of TME and improve the therapeutic effect,the strategies of tumor-targeted,combination therapy and stimuli-responsive drug delivery systems (DDSs)have been proposed and summarized in Figure 2 and Table 1.Aliasger K Salem’s and Chun Li’s study provided the proof-of-concept that the combination of PTX-loaded nanoparticles with the antiangiogenic molecular inhibitor BIBF 1120 (BIBF) and sonic hedgehog inhibitor cyclopamine (CPA) has marked inhibition of tumor progression in loss-of-function LOF p53 tumors and pancreatic cancer respectively [21,22].Synergistically inhibiting progression of established tumor via the combination of PTX-mediated chemotherapy with immunotherapy,photodynamic therapy,photothermal therapy with the help of nanotechnology has been approved [23-25].In order to achieve precise delivery of PTX,augmenting the intrinsic enhanced permeability and retention effect with an intrinsic vascular modulator such as nitric oxide,conjugated with targeting ligand such as K237 peptide (tumor neovessles-targetable ligands),Ep23 aptamer (against epithelial cell adhesion molecule,and Herceptin (for HER2-positive breast cancer,synthesizing a biomimetic macrophage-membranecoated nanoparticle with the inflammatory tumorhoming ability and precoating transferrin-modified nanoparticles with protein coronas derived from healthy mice significantly enhanced active targeting capacities were developed in recent years [26-30].Chen Jiang,Xiangliang Yang and Jin Sun et al.designed and synthesized PTX-loaded and redox-responsive DDSs containing disulfide bond (S-S) bond and double selenium bond (Se-Se) for significant inhibition of tumor cell growth with minimal side effect [31-33].Because of higher reactive oxygen species (ROS)levels in tumor than normal organs,thioketal (TK)linker was utilize in DDSs which exhibited specifically release of PTX avoiding cytotoxicity to normal cells[34,35].Additionally,pH-sensitive DDSs were constructed to improve PTX delivery for enhanced cancer therapy based on pH difference between tumor microenvironment (pH 6.5) and normal tissue or blood(pH 7.4) [36-38].Moreover,dual stimuli-responsive DDSs were designed for PTX delivery to fulfill the programmable on-demand drug release both extracellularly and intracellularly in cancer cells [39-41].In short,PTXs nano-formulations have shown great potential for tumor treatment.

Figure 2 Schematic illustration of PTX-loaded nanoparticles for enhanced cancer therapy.(A) Hypoxiaactivated self-immolative paclitaxel prodrug (PTX2-Azo) was synthesized and combined with PDT to prepare lightboosted PTX nanoparticle (Ce6/PTX2-Azo NP) [24].(B) PTX @ PCL-PEG-Herceptin passively targets tumors via the EPR effect and actively target HER2 receptors [28].(C) (a) A simple glutathione-responsive turn-on theranostic nanoparticle consisting of adisulfide-bond-linked hydroxyethyl starch paclitaxel conjugate (HES-SS-PTX) and a near-infrared cyanine fluorophore DiR [31].(b) ROS-sensitive linkage-bridged diblock copolymer-based nanocarriers TK-NPCe6&PTX for on-demand drug delivery by photo-induced PEG deshielding [34].(c) pH multistage responsive PTX- and CUR-loaded PPBV micelles with the PEG detachment and charge-switching function for enhanced cellular uptake and tumor penetration [38].PTX,paclitaxel;PDT,photodynamic therapy;TK,thioketal;ROS,reactive oxygen species;EPR,enhanced permeability and retention.

Table 1 Different strategies were developed for improved PTX delivery

ATO

ATO has been clinically approved as an effective drug for the treatment of acute promyelocytic leukemia,which arouses research interest in its anti-tumor activity in other malignant tumors and solid tumors [4,42,43].Unfortunately,ATO is not as effective as acute promyelocytic leukemia for solid tumors [44].In order to achieve effective therapy,the dose of ATO required for solid tumor treatment will increase,accompanied by increased toxicity.To address this problem,researchers have begun to try to deliver ATO through nanotechnology and have published nearly one hundred scientific papers in this field [45].These strategies were summarized in Figure 3 and Table 2.One strategy is to prepare ATO-loaded nanoparticles by taking advantage of weak acid transport properties and the fact that arsenite forms insoluble transitional metal (e.g.,Ni2+,Co2+,Cu2+,Zn2+,Mn2+,and Fe2+) compounds [46-51].In 2006,Thomas V.OHalloran and co-workers synthesized a series of liposome-encapsulated insoluble transitional metal arsenite compounds,which were stable in physiological situations but could release drug at low pH [48].Based on similar principles,Jinhao Gao’s group reported ATO-based smart multifunctional DDS MnAsOx@SiO2.Acidic stimuli triggered the simultaneous release of manganese ions (Mn2+) and ATO,which dramatically increased the T1 signal,enabled real-time visualization and monitoring of ATO release and delivery,and efficiently delivers ATO to treat tumors to inhibited the growth of solid tumors without adverse side effects [46].Another strategy is to apply polymer as the carrier for ATO delivery [52-56].Yourong Duan et al applied mPEG-PLGA-PLL to load ATO for hepatocellular carcinoma [14].Haifa Shen et al prepared bone-targeting nanoparticle BTNP to codeliver ATO and decitabine through PLGA-cholesterol core [15].Moreover,arsenic-sulfur (As-S) bond was employed to prepare ATO-loaded nanoparticles [57,58].Weibo Cai et al found that thiolated MSNs had a macroscopic arsenic loading capacity of 20 mg of ATO per gram of MSN due to the specific binding between trivalent arsenic and nanoparticle thiol surface modification [57].Weihong Tan et al loaded ATO onto folate (FA)-labeled human serum albumin pretreated with glutathione (GSH) based on the low pH- and GSHsensitive arsenic-sulfur bond,and indicated that the nanodrug significantly improved therapeutic efficacy and alleviated side effects of ATO in chronic myeloid leukemia treatment [58].In addition,erythrocyte membrane,human serum albumin (HSA) and inorganic nanoparticles can also be used as carriers to deliver ATO[59-62].

CPT

CPT is a natural hydrophobic alkaloid and a valuable compound for cancer chemotherapy as a topoisomerase I inhibitor and exhibits cytotoxic activity towards several cancer cells.However,CPT is highly hydrophobic and non-specific,which hinders its clinical application.In the past few decades,stimuli-responsive DDSs have been developed in response to specific stimuli (e.g.,pH,GSH,enzyme,ATP,hypoxia,temperature,magnetic field,light) to improve the therapeutic efficacy and were summarized in Figure 4 and Table 3.TK was employed to designed and synthesized CPT-loaded nanoparticles respond to the high ROS levels in cancer cells [63-66].Xiaoyuan Chen’s group developed a ROS-activatable heterodimeric prodrug of a synergistic drug pair(chemo-drug CPT and photosensitizer HPPH) via a TK linker for tumor-selective imaging and therapy [66].As GSH content in the cytosol and cell nucleus (2 to 10 mM) is about ～1000-fold higher than that in body fluids and extracellular matrices (2-10 μM),redox-responsive nanoparticles containing bonds of S-S and Se-Se begin to thrive [67-72].Karen L.Wooley et al found that increases in degrees of CPT conjugation decreased aqueous accessibility and reduced glutathione-triggered release and demonstrated the impact of hydrophobic drug conjugation on the complex interplay between the level of drug loading on the polymer nanoparticle physicochemical characteristics and the resulting access of molecular stimuli to the internal compartments and subsequent triggered drug release [67].Moreover,weakly acidic TME is also used to design pH-sensitive CPT-loaded nanoparticles [73-76].Vladimír Sedlařík et al prepared CPT-loaded nanoparticles with tunable surface charges which increase from around -10 mV in a normal physiological condition (pH 7.4) to +40 mV under acidic tumor environments to achieve better drug delivery [75].Yonghong Liao et al reported hyaluronan stacked self-assembling nanoparticles with intracellular transformations of size and surface charge due to the presence of hyaluronidase and acidic pH in the endosomes/lysosomes,resulting in improved tumor penetration,internalization,and intracellular drug release [76].In addition,ultrasound,laser and enzyme were applied to trigger CPT release for improved drug delivery and enhanced antitumor efficacy [77-82].

Figure 3 Schematic illustration of ATO-loaded nanoparticles for enhanced cancer therapy.(A) Depiction of ZnAs@SiO2 NPs synthesis by a “one-pot” reverse emulsification approach [47].(B) Schematic illustration of the designed structure of bone-targeting nanoparticles composed of alendronate-conjugated PEG-DSPE for bone targeting,and the PLGA-cholesterol core in-corporated decitabine and ATO together [53].(C) Radioarsenic-labeled thiolated mesoporous silica for developing PET-guided ATO-based drug delivery strategy was prepared based on the specific binding between *As (III) and thiol group surface modifications of the silica nanoparticles [57].(D) A kind of hybrid nanovehicles consisting of PAA modified mesoporous silica core and an angiopep-2-modified liposomal shell to achieve targeted delivery and on-demand release of ATO for brain glioma therapy [62].ATO,arsenical trioxide.

Table 2 Different strategies were developed for improved ATO delivery

Conclusion and outlook

Combination ancient effective ingredients extracted from TCM and advanced nanotechnology have great potential for improved cancer therapy.Different research groups worldwide have engineered effective ingredients such as PTX,ATO and CPT into multifunctional nano DDSs for improved drug delivery.This strategy may offer a novel approach for the development of new nanomedicines with high translation potentials and wide clinical application.However,there are still some challenges for accelerating the development of TCM effective ingredients-engineered nanomedicines to consider.Firstly,there is still a great library of TCM effective ingredients that have not been investigated for cancer therapy. A better understanding of their pharmacological mechanism,physicochemical properties and quality evaluation is extremely important for screening proper TCM effective ingredients as anticancer drugs or a supplementary means for enhancing conventional cancer therapy.Secondly,TCM effective ingredients usually have the characteristics of multiple targets,which is like a coin with two sides and has positive (pharmacological activity) and negative(toxic and side effects) effects.How to effectively utilize or amplify its pharmacological activity and reduce or eliminate its side effects is a difficult problem.In addition,although TCM has been used for thousands of years,pharmacokinetics,distribution,metabolism and safety in vivo of TCM effective ingredients should still be systematically investigated.The interactions among TCM effective ingredients,nanocarriers,other components of a composite nanomedicine and complex physiological and pathological microenvironment in the body may influence the behaviors in vivo as well.Moreover,we should also take advantages of novel nanotechnology for improved TCM effective ingredients delivery and broadened range of biomedical applications.Finally,the large-scale industrial production and quality control of nanomedicines are also challenging issues.It is expected that these TCM effective ingredients will benefit the further advancement of cancer nanomedicines with superior efficacy and safety by addressing these issues.

Figure 4 Schematic illustration of CPT-loaded stimulus-responsive nanoparticles for enhanced cancer therapy.(A) Scheme of prodrug HRC-encapsulated by F127 (HRC@F127nanoparticles),which is activated by ROS,for cancer imaging and synergetic chemo- and photodynamic treatment [66].(B) Supramolecular polymers constructed from the host-guest complexations between CPT and β-CD orthogonally self-assemble into SNPs,which rapidly dissociated arising from the cleavage of disulfide bond (S-S) by intracellular GSH [70].(C) Construction of chargeconversional drug self-delivery systems for better targeted cancer therapy [75].(D) Schematic illustration of selfassembly of PCF-MBs with porphyrin grafted lipid and camptothecin-floxuridine conjugate,which can be converted into PCF-NPs by ultrasound targeted MB destruction and (b) PCF-MBs mediated chemo-photodynamic combination therapy with the assistance of UTMD technique [77].(E) Schematic illustration of the switchable phototheranostic nanoparticles for light-controlled pulsatile drug release in cancer photothermal-chemotherapy,which was navigated by photoacoustic imaging [79].(F) Schematic illustration of enzyme-triggered morphology transformation and autocatalytic growth of nanofibers [82].ROS,reactive oxygen species.

Table 3 Stimuli-responsive DDSs were designed for improved CPT delivery

Table 3 Stimuli-responsive DDSs were designed for improved CPT delivery (continued)