Jiagen Li,Xinming Li,Pengwei Xie,Peng Liu

State Key Laboratory of Applied Organic Chemistry,College of Chemistry and Chemical Engineering,Lanzhou University,Lanzhou,730000,China

Keywords:

Drug self-delivery system

Regulated drug release

Doxorubicin-doxorubicin dimer

Acid-triggered release

Mixed nanoparticles

A B S T R A C T

A mixed drug self-delivery system(DSDS)with high drug content(＞50%)was developed to regulate pH-triggered drug release,based on two doxorubicin(DOX)-DOX dimmers:D-DOXADHand D-DOXcarconjugated with acid-labile dynamic covalent bonds(hydrazone and carbamate,respectively)and stabilized with PEGylated D-DOXADH(D-DOXADH-PEG).Owing to the different stability of the dynamic covalent bonds in the two dimers and the noncovalent interaction between them,pH-triggered drug release could be easily regulated by adjusting the feeding ratios of the two DOX-DOX dimers in the mixed DSDS.Similar in vitro cellular toxicity was achieved with the mixed DSDS nanoparticles prepared with different feeding ratios.The mixed DSDS nanoparticles had a similar DOX content and diameter but different drug releasing rates.The MTT assays revealed that a high anti-tumor efficacy could be achieved with the slowrelease mixed DSDS nanoparticles.

1.Introduction

Doxorubicin(DOX),a broad-spectrum chemotherapeutic agent,has been widely used for cancer treatment[1].However,it usually presents severe toxic side effects on normal cells and tissues because of its non-specificity[2].Therefore,tumor specific drug delivery systems(DDSs)have been investigated intensely for DOX to improve its anti-tumor efficacy and minimize its toxic side effects on normal cells and tissues,in the form of DOX-loaded nanocarriers via non-covalent interactions(such as electrostatic or hydrophobic interaction,π-π stacking,or hydrogen bonds)[3],or as prodrugs via acid-labile or reduction-cleavable dynamic covalent bonds[4].

Owing to the acid-labile or reduction-cleavable dynamic covalent conjugations in the prodrugs,which can be cleaved to release the drug upon stimuli of acid or a high reductant level in the tumor cells,minimized premature drug leakage can be achieved to avoid the toxic side effects.Therefore,these prodrugs have attracted increasing attention in the last decades[4].For the DDSs,the particle shape and size and surface property mainly affect their internalization into the tumor cells[5],whereas the drug content and drug releasing rate are the main factors determining antitumor efficacy.Particularly,after cellular uptake,a low intracellular drug concentration is usually achieved with the DDSs with low drug content.Moreover,the low intracellular drug concentration can barely kill the tumor cells but might cause multidrug resistance[6].

Compared with the high anti-tumor efficacy of DOX released from the acid-labile prodrugs,the low anti-tumor efficacy has been achieved for the reduction-cleavable prodrugs,because the released drug is usually a thiolated derivative of DOX[7].Furthermore,in most of the reported tumor-specific polymer prodrugs,the chemotherapeutic agents such as DOX are usually conjugated onto the side groups or end groups of the polymer chains[8];thus,it is a pity that only a low drug content could be obtained.Therefore,the development of an acid-labile prodrug with high drug content but low premature drug leakage is urgent for cancer treatment.

Most recently,drug self-delivery systems(DSDSs),also known as cargo-free nanomedicines,have drawn much attention for cancer treatment,achieving intracellular delivery on their own without any nanocarriers[9].With the desired high drug content similar to that in the drug nanocrystals[10],the dimer prodrugs exhibit a lower premature drug leakage than the pure chemotherapeutics owing to their lower solubility.However,the drug releasing rate is mainly determined by the acid-labile dynamic covalent bond in the dimers,in addition to the diameters of the DSDSs[11,12].

Besides the high drug content,the drug releasing rate is another key factor in controlling the anti-tumor efficacy of the DDSs[13].Till date,different strategies have been established to modulate the drug releasing rate such as dual-modal drug-loading[14,15]or tuning the surface textures of the drug-carriers[16].As an ideal acid-labile DSDS with high drug content,the chemotherapeutic drug is expected to be released at a desired rate to kill the cancer cells.

In the present study,novel DSDS nanoparticles with high drug content(＞50%)were developed to regulate the pH-triggered drug release,by stabilizing two DOX-DOX dimers conjugated with acidlabile dynamic covalent bonds:D-DOXADHvia a hydrazone bond with a rapid DOX releasing rate and D-DOXcarvia a carbamate bond with a slow DOX releasing rate,with the PEGylated D-DOXADH(DDOXADH-PEG)as an emulsifier(Scheme 1).Different DOX releasing rates,despite similar DOX content and diameter,were achieved by adjusting the feeding ratios of the two DOX-DOX dimers in the proposed DSDS.In addition,all the proposed DSDSs exhibited the enhanced anti-tumor efficacy on HepG2 cells in comparison to that with the free DOX.

2.Experimental

2.1.Reagents and chemicals

mPEG-COOH(95%,weight-average molecular weight=2000 Da)was purchased from Shanghai Biotech Co.,Ltd.(Shanghai,China).Doxorubicin hydrochloride(DOX·HCl,99.4%)was purchased from Beijing Huafang United Technology Co.,Ltd.(Beijing,China).1,5-Pentanediol(98%)was obtained from Chengdu Chron Chemicals Co.,Ltd.(Chengdu,China).4-Nitrophenyl chloroformate(98%)was obtained from J&K Chemical Ltd.(Beijing,China).Adipic acid dihydrazide (ADH, 98%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (99%),1-hydroxybenzotriazole(99%),and 4-dimethylaminopyridine(99%)were purchased from J&K Chemical Ltd.(Beijing,China).All other reagents and solvents were of analytical grade and used without further purification.Double distilled water was used throughout the experiments.

2.2.Preparation and characterization of mixed DSDS nanoparticles

D-DOXcarvia an acid-labile carbamate linker with a DOX content of 86.59% was synthesized by conjugating two DOX molecules with one bis(4-nitrophenyl)pentane-1,5-diyl bis(carbonate)molecule as reported previously[11].1H NMR(400 MHz,dimethyl sulfoxide DMSO-d6,Fig.S1):δ1.54-1.40(f,6.00H),4.03-3.76(a+e,10.02H),7.66-7.53(c,2.01H),7.96-7.81(b+d,4.00H).

D-DOXADHvia an acid-labile hydrazone linker with a DOX content of 88.76% was synthesized by conjugating DOX molecules with ADH as described previously[12].1H NMR(400 MHz,DMSO-d6,Fig.S2):δ4.02-3.89(a,6.00H),2.22-2.08(b,4.01H),1.62-1.48(c,3.96H).The D-DOXADH-PEG with a DOX content of 33.92% was synthesized as an emulsifier by the PEGylation of D-DOXADHwith mPEG-COOH[12].1HNMR(400MHz,DMSO-d6,Fig.S3):δ3.63-3.44(b,176.00H),4.05-3.90(a,5.95H).

Scheme 1.Preparation of the mixed drug self-delivery system(DSDS)nanoparticles.ADH:adipic acid dihydrazide;DOX:doxorubicin.

Then,the mixed DSDS nanoparticles(D-DOXmixNPs)were prepared by emulsifying the two DOX-DOX dimers,D-DOXcarand DDOXADH,at different feeding ratios,with D-DOXADH-PEG as the emulsifier(Table 1).Taking D-DOXmix-3 as an example,D-DOXcar(5.00mg),D-DOXADH(5.00mg),and D-DOXADH-PEG(10.00mg)were dissolved in 5.00mL of DMSO.Then the solution was added drop-bydrop to 50 mL water with violent stirring.After stirring for 5 min,the D-DOXmix-3 NPs were collected by centrifugation(10,000 r/min for 5 min),washed with water,and dried in vacuum at 40°C.

Table 1Doxorubicin(DOX)content of the mixed drug self-delivery system(DSDS)nanoparticles(D-DOXmixNPs)prepared with different feeding ratios.

Through sampling with their aqueous dispersions,the particle morphology and hydrodynamic diameter of the obtained D-DOXmixNPs were characterized using transmission electron microscope(TEM,Talos F200c,Thermo Fisher Scientific,Waltham,MA,USA)and dynamical light scattering(DLS,BI-200SM,Brookhaven Instruments Corp.,Holtsville,NY,USA),respectively.

The DOX content of the obtained DOX-DOX dimer and D-DOXmixNPs was determined in a 0.02 mg/mL DMSO solution with a UV-vis spectrometer(Lambda 35,PerkinElmer,Inc.Shelton,CT,USA)with absorbance at 480 nm,and calculations based on the standard curve of DOX·HCl in DMSO[11,12].

2.3.Acid-triggered drug release

Ten milliliters of the dispersion of the D-DOXmixNPs(0.20 mg/mL)in different buffer solutions(phosphate-buffered saline(PBS)at pH 7.4 or acetate-buffered solution(ABS)at pH 5.0)was transferred into a dialysis bag(molecular weight cut-off of 1 kD).The dialysis bag was then immersed in the corresponding buffer solution(100 mL).After a predetermined time,5.0 mL of the dialysate was removed to measure the concentration of the released DOX using the UV-vis spectrometer(Lambda 35,PerkinElmer,Inc.Shelton,CT,USA)at an absorbance wavelength of 480 nm.Fresh solution(5.0 mL)was added to keep the total volume constant.

2.4.In vitro cellular toxicity and uptake

HepG2 cells were used as model tumor cells after cultivation on 96-well plates(1.0× 105cells/well)in 100μL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and incubation in atmospheric humidity(5% CO2,95% air,37°C)for 24 h.

The in vitro cytotoxicity of the proposed D-DOXmixNPs was evaluated via the MTT assay.After the HepG2 cells were incubated with the D-DOXmixNPs or free DOX at different concentrations for 24 h,20.0μL of 5.0 mg/mL MTT was added into each well,for further incubation of 4 h.Then,the MTT-containing medium was removed and replaced with 150μL of DMSO.After the crystal substances were dissolved for 20 min in each well,the absorbance of the solution was measured at 490 nm on a microplate reader.

The in vitro cellular uptake of the D-DOXmix-3 NPs was examined using confocal laser scanning microscopy(DMI 4000B,Leica,Wetzlar,Germany)after incubating the HepG2 cells with 15μg/mL D-DOXmix-3 NPs for 24 h.The cells were washed with the culture solution,fixed with 4% paraformaldehyde,and dyed with 4′,6-diamidino-2-phenylindole(DAPI).The fluorescence was analyzed at 480 nm for DOX and 461 nm for DAPI.

2.5.Statistical analysis

All data are presented as mean±standard deviation.The statistical analyses were performed with the one-way ANOVA and the Student's t-test was used for two-group comparisons(SPSS software,version 16.0,SPSS Inc.;Chicago,IL,USA).Statistical significance was set at P＜0.05.

3.Results and discussion

3.1.Preparation and characterization of mixed DSDS nanoparticles

Because of the hydrophobicity of the two dimers,as well as the anthracene rings,they could precipitate in water via noncovalent interactions,such as the hydrophobic interaction and π-π stacking interaction.The D-DOXmixNPs were prepared as mixed DSDS NPs by stabilizing the slow-releasing D-DOXcarand the fast-releasing DDOXADHat different feeding ratios with D-DOXADH-PEG as the emulsifier(Scheme 1).

The mean hydrodynamic diameters(Dh)of the D-DOXmixNPs were less than 200 nm(Fig.1),indicating that the D-DOXmixNPs could be passively targeted to tumor sites via the enhanced permeability and retention effect.With an increase in the feeding ratio of D-DOXcarwith D-DOXcar:D-DOXADH:D-DOXADH-PEG from 0:10:10 to 2.5:7.5:10,5:5:10,7.5:2.5:10,and 10:0:10(mg/mg/mg),it was clear that the Dhdecreased from 194 nm for the D-DOXmix-1 NPs to 187 nm for the D-DOXmix-2 NPs,180 nm for the D-DOXmix-3 NPs,161 nm for the D-DOXmix-4 NPs,and finally,125 nm for the DDOXmix-5 NPs(Fig.1).This might be owing to the different hydrophilic-hydrophobic properties of the two DOX-DOX dimers.D-DOXADHwas synthesized by conjugating DOX on its carbonyl group with ADH and the amino group in DOX was retained;therefore,it should have a higher hydrophilic property than the DDOXcarconjugated on the amino group in DOX.

Fig.1.Typical hydrodynamic diameter distributions of the mixed drug self-delivery system nanoparticles(D-DOXmixNPs)in pH 7.4 phosphate-buffered saline.

In the TEM analysis,all the mixed DSDS NPs showed a near spherical shape.Similar to the results with the DLS,the particle size decreased from 90 nm for the D-DOXmix-1 NPs(Fig.2A)to 78 nm for the D-DOXmix-2 NPs(Fig.2B),65 nm for the D-DOXmix-3 NPs(Fig.2C),60 nm for the D-DOXmix-4 NPs(Fig.2D),and finally,43 nm for the D-DOXmix-5 NPs(Fig.2E),with the increase in the feeding ratio of D-DOXcar.The Dhfrom the DLS analysis was much higher than that of the particle size from TEM analysis owing to surface PEG brushes in the D-DOXADH-PEG,as well as the solvation of the DOX-DOX dimers.

Fig.2.Transmission electron microscopy images of(A)the D-DOXmix-1 NPs,(B)D-DOXmix-2 NPs,(C)D-DOXmix-3 NPs,(D)D-DOXmix-4 NPs,(E)D-DOXmix-5 NPs in pH 7.4 phosphate-buffered saline,and(F)the D-DOXmix-3 NPs after treating with pH 5.0 acetate-buffered solution for 36 h.

The DOX content of the D-DOXmixNPs prepared with different feeding ratios was determined using a UV-vis spectrometer and is listed in Table 1.With the different D-DOXcar:D-DOXADH:D-DOXADH-PEG feeding ratios,the DOX content of the D-DOXmixNPs was in the range of 56%-60%.In addition,the determined DOX content was close to the theoretical values calculated from the corresponding DOX content of the components at certain feeding ratios,demonstrating a high yield in the emulsification.

3.2.pH-triggered drug release profiles

The acid-triggered drug release profiles of the D-DOXmixNPs were evaluated in the different releasing media,pH 7.4 PBS and pH 5.0 ABS,mimicking the normal physiological media and tumor intracellular microenvironment,respectively.The effect of the emulsifier D-DOXADH-PEG on the drug release behavior was first investigated by comparing the drug release profiles of the pure DDOXcarNPs and D-DOXADHNPs with the emulsified ones(D-DOX-mix-3a NPs and D-DOXmix-3b NPs).As shown in Fig.3A,the DDOXmix-3b NPs showed a much lower drug leakage of 6.85% than the 13.30% of pure D-DOXADHNPs in pH 7.4 PBS.This was because the D-DOXADH-PEG in the D-DOXmix-3b NPs could be slightly cleaved to release DOX and DOX-PEG;the former could diffuse through the dialysis bag and be determined in the dialysate,whereas the latter remained in the dialysis bag owing to its higher molecular weight.

Fig.3.Acid-triggered drug release profiles of the D-DOXmixNPs:effects of(A)the emulsifier D-DOXADH-PEG and(B)the feeding ratios of the D-DOXcarand D-DOXADH.

Regarding the D-DOXcardimer,the D-DOXmix-3a NPs showed a slightly higher drug leakage of 2.41% than the 2.11% of pure DDOXcarNPs.The carbamate bond in the D-DOXcardimer is a dynamic covalent bond with higher stability than that of the hydrazone bond in the D-DOXADHdimer[17].Therefore,it was used for the intracellular pH-triggered slow DOX release to eradicate any residual or latent cancer cells that lead to the recurrence of tumor[11].However,the D-DOXADH-PEG in the D-DOXmix-3a NPs could be cleaved to release DOX.

In the pH 5.0 ABS mimicking the tumor intracellular microenvironment,the D-DOXmix-3b NPs gave a lower drug release of 66.75% than the pure D-DOXADHNPs,whereas the D-DOXmix-3a NPs showed a slightly higher drug release of 5.40% than the pure DDOXcarNPs,similar to the drug release profiles in the pH 7.4 PBS mimicking the normal physiological media.This demonstrated that the drug release performance was mainly determined by the DOXDOX dimers.To be precise,it was dependent on the dynamic covalent conjugating bonds in the DOX-DOX dimers.

Next,the effect of the feeding ratios of D-DOXcarand D-DOXADHon the acid-triggered drug release from the D-DOXmixNPs was investigated.The results are shown in Fig.3B.With increasing feeding ratio of the slow-release dimer D-DOXcar,the drug leakage at pH 7.4 and drug release at pH 5.0 decreased noticeably.The results demonstrated that the drug release rate could be efficiently modulated by adjusting the feeding ratio of the slow-releasing dimer D-DOXcar.Comparison of the drug release profiles of the DDOXmix-3b and D-DOXmix-3 NPs,both possessing the same DDOXADH:D-DOXADH-PEG feeding ratio of 5:10(mg/mg),showed that the D-DOXmix-3 NPs with a higher drug content(59.50%)had a lower drug release amount(drug content×cumulative drug release)of 0.25 mg/mg in pH 5.0 ABS within 36 h,whereas the DDOXmix-3b NPs with a lower drug content(52.61%)showed a higher drug release amount of 0.35 mg/mg.These results indicate that the regulated drug release rate was ascribed to not only the slow drug release rate from the slow-releasing dimer D-DOXcar,but also the inter-molecular noncovalent interaction between the DOXDOX dimers.Owing to the noncovalent interaction,the cleavage of the fast-releasing dimer D-DOXADHwas reduced by the slowreleasing dimer D-DOXcar.These two factors,both the slow release rate from D-DOXcarand the restricted release rate from DDOXADHowing to the noncovalent interaction with the D-DOXcar,determined the drug release rate from the proposed mixed DSDSs.

Taking the D-DOXmix-3 NPs as an example,which were prepared with a D-DOXcar:D-DOXADH:D-DOXADH-PEG feeding ratio of 5:5:10(mg/mg/mg),they remained in a spherical shape with a small size after treatment with pH 5.0 ABS for 36 h,without breaking into small pieces that could be produced during the potential disintegration of the D-DOXmix-3 NPs(Fig.2F).Based on this phenomenon,the drug releasing mechanism could be speculated as follows:first,the H+cations attacked the hydrazone bond on the surface DDOXADH-PEG layer to produce DOX and DOX-PEG and DOX was released into the releasing medium.Then,the H+cations diffused slowly into the hydrophobic cores of the D-DOXmix-3 NPs and cleaved the acid-labile dynamic covalent bonds in the dimers.The carbamate conjugation in the D-DOXcaris more stable than the hydrazone bond in the D-DOXADH;therefore,the D-DOXADHwas easily cleaved in a restricted mode owing to the noncovalent interaction with the D-DOXcar.Thus,the remaining D-DOXcardimers were adsorbed on the nanoparticles with the emulsifying effect of the DOX-PEG.As the release time was prolonged,more DDOXADHdimers in the hydrophobic cores were cleaved off,a shell layer of the slow-releasing dimer D-DOXcarwas formed;as a result,a more obvious impediment effect was produced.The drug releasing rate became slower and slower with the increase in time(Fig.3B).

The drug release data were then fitted with Higuchi and Korsmeyer-Peppas equations to further understand the drug releasing mechanism(Fig.S4).As shown in Table 2,the correlation coefficients from the Higuchi equation were higher than those from the Korsmeyer-Peppas equation in most cases,indicating a better fit with the Higuchi equation of the diffusion mechanism based on Fick's law for water-soluble drugs[18],namely,the released DOX after the cleavage of the dimers in the present study.

Table 2Fitted parameters with the Higuchi and Korsmeyer-Peppas models.

3.3.In vitro cellular toxicity and intracellular distribution

The cellular toxicity of the proposed D-DOXmixNPs on the HepG2 cancer cell line was evaluated in vitro after incubation for 24 h,in comparison with the free DOX at different dosages.Similar to that with the free DOX,the cell viability of HepG2 cells decreased with increased dosage of the three kinds of D-DOXmixNPs with similar DOX content from 2.5 to 20μg/mL(Fig.4).However,the cell viabilities after incubation with the D-DOXmixNPs were higher than those with the free DOX at the same dosage.At each dosage,the cell viability decreased from the D-DOXmix-1 NPs to the D-DOXmix-5 NPs.

Fig.4.In vitro cell viability of HepG2 cells against the D-DOXmixNPs and free DOX after incubation for 24 h(＊P＜0.05).

At a dosage of 20μg/mL,the cell viability was in the range of 52%-58% for the D-DOXmixNPs,whereas it was only approximately 39% after incubation with free DOX.For the D-DOXmix-1 NPs,which were prepared with a D-DOXADH:D-DOXADH-PEG feeding ratio of 10:10(mg/mg)and showed the fastest drug releasing rate among the three samples,the cumulative drug release was 84.5% in the simulated tumor intracellular microenvironment within 24 h(Fig.3B).After 24 h of incubation,the actual amount of released DOX was calculated to be 9.6μg/mL,estimated as the multiplication of the DOX content,dosage,and cumulative drug release in 24 h of the D-DOXmix-1 NPs.The D-DOXmix-5 NPs,prepared with a D-DOX-car:D-DOXADH-PEG feeding ratio of 10:10(mg/mg),showed the slowest drug release rate among the three samples,only 4.20% at pH 5.0 within 24 h.The actual amount of released DOX was calculated as 0.5μg/mL.As for the D-DOXmix-3 NPs prepared with a D-DOXcar:D-DOXADH:D-DOXADH-PEG feeding ratio of 5:5:10(mg/mg/mg),a moderate drug release rate was achieved,with a cumulative release of 39.16% at pH 5.0 within 24 h.The actual amount of released DOX was calculated as 4.7μg/mL.D-DOXADH-PEG could be cleaved to release DOX and DOX-PEG intracellular the tumor cells.The D-DOXADH-PEG content in the D-DOXmixNPs was approximately 50%.The actual amount of released DOX was 1.7μg/mL in the DOX-PEG if the D-DOXADH-PEG was completely cleaved off.Considering the complete release of the DOX and DOX-PEG from the D-DOXmixNPs,the actual total amount of released DOX was 11.3,6.4,and 2.2μg/mL from the D-DOXmix-1 NPs,D-DOXmix-3 NPs,and D-DOXmix-5 NPs within 24 h,respectively.With the decline in actual total amount of released DOX,increased cytotoxicity was achieved for the three DSDS nanoparticles,demonstrating that the higher cytotoxicity resulted from the slower drug release rate[11].This was probably caused by the more complete inhibition of the P-gp drug efflux pump with the slow-releasing DSDS nanoparticles.Furthermore,the cell viability of the D-DOXmix-5 NPs was much lower than that of the free DOX at 2.5μg/mL,indicating an enhanced anti-tumor efficacy of the slow-release mixed DSDS nanoparticles.However,the D-DOXmix-1 NPs showed the lowest actual cytotoxicity,maybe owing to the P-gp drug efflux pump being similar to that of the free drug[19],because of its faster drug releasing rate.

Finally,the in vitro cellular uptake and intracellular distribution of the D-DOXmix-3 NPs were assessed using the confocal laser scanning microscopy technique,after incubation of the HepG2 cells with the D-DOXmix-3 NPs(15μg/mL)for 24 h.Obvious cell shrinkage with less cytoplasm mass could be seen in the bright field image of the HepG2 cells after incubation with the D-DOXmix-3 NPs(Fig.5A),demonstrating the cytotoxic effect mediated through apoptotic cell death[20].As seen in Fig.5B,the blue fluorescence regions are the cell nuclei stained with DAPI,whereas the red fluorescence positions in Fig.5C show the intracellular distribution of DOX.In the merged image(Fig.5D),the red fluorescence positions are completely covered by the blue fluorescence regions,indicating the nuclear accumulation of the released drug in the HepG2 cells[21].

Fig.5.Confocal laser scanning microscopy images of the HepG2 cells after incubating with the D-DOXmix-3 NPs(15 μg/mL)for 24 h:(A)bright field,(B)4′,6-diamidino-2-phenylindole,(C)DOX,and(D)the merged image.

4.Conclusions

In summary,a facile strategy was established to regulate the pH-triggered drug release rate,by fabricating a mixed DSDS with high drug content(＞50%)with DOX-DOX dimers(a fast-releasing dimer D-DOXADHand a slow-releasing dimer D-DOXcar),stabilized with DDOXADH-PEG.The in vitro drug release profiles showed that the drug release rate could be efficiently modulated by adjusting the feeding ratio of the dimers.However,an increased cytotoxicity was achieved for the proposed D-DOXmixNPs with similar DOX content but slower DOX releasing rate in the MTT assays.The results further demonstrated that an enhanced anti-tumor efficacy could be achieved with the slow-release mixed DSDS NPs.Such understanding would provide a new insight into designing a promising nanoformulation with high drug content,excellent acid-triggered drug release,and enhanced antitumor efficacy for future tumor treatment.

CRediT author statement

Jiagen Li:Investigation,Visualization,Data curation;Xinming Li:Investigation,Visualization;Pengwei Xie:Investigation;Peng Liu:Conceptualization,Methodology,Investigation,Writing-Reviewing and Editing,Supervision.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2021.03.001.