Supporting Information Paclitaxel prodrug based mixed micelles for tumor targeted chemotherapy

a Department of Experimental Center, Henan Institute of Science and Technology, Xinxiang, Henan 453003, P. R. China b Department of Pharmacy, Xinxiang Central Hospital, Xinxiang, Henan 453000, P. R. China c Nanjing Research Center, Jiangsu Chiatai Tianqing Pharmaceutical Co. Ltd, Nanjing, Jiangsu 210042, P. R. China d College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, P. R. China


Introduction
Until now, drug delivery in oncology remains of particular interest owing to the relatively narrow therapeutic window of many antineoplastic agents.The unfavorable effects, particularly the untargeted side effects as well as poor water solubility, signicantly limit their further application in clinical practice.To address these dilemmas, micelles as a preferable drug delivery system (DDS) based on various types of polymeric materials have been proposed. 1,2Micelles have raised a special interest as nano-sized DDSs not only because they provide an increased solubility and stability of hydrophobic drugs, 3,4 but also due to their superior advantages versus the free drug both in vitro and in vivo. 5Micelles with a size between 20 and 200 nm are large enough to prevent the premature elimination via glomerular ltration and sufficiently small to pass through certain blood vessels. 6Moreover, they are capable of improving the cellular uptake of encapsulated drugs as well as granting an alternative route of internalization (endosomes).The latter application is of vital importance in overcoming the multi-drug resistance (MDR) that is responsible for the poor performance of many anti-neoplastic agents. 7wever, most of the currently available micelles have usually succumbed to their poor drug loading capacity as well as uncontrollable drug leakage during the delivery process.One of the most studied approaches to compensate such aws is to employ the drug molecules as the hydrophobic block of the micelle and as the therapeutic moiety, 8 which is also referred as prodrug micelles (PMs).The prodrugs, by denition, are the derivatives of drugs that can be metabolized or activated in the body or cell to release or regenerate the active drugs. 9,10In the past decades, numerous research efforts have concentrated on conjugating antitumor drugs with a wide spectrum of low-and high-molecular-weight carriers including natural product and synthetic polymers. 11By selectively choosing the hydrophilic and hydrophobic parts of the prodrug, they can automatically ock together in an aqueous solution above the critical micelle concentration (CMC), resulting in the formation of PMs. 12 The hydrophobic part of the drug can thereby gather to form the core of the micelle, in which the drug molecules can be entrapped by an outer hydrophilic shell, which isolates the encapsulated drug from the external medium that allows for the effective protection from the hostile in vivo environments. 13he incorporation of various functionalities such as a targeting ligand to seek out the overexpressed surface receptors on the diseased cells provides a more efficient and controlled therapy approach. 14However, the concomitant incorporation of multiple functionalities in a single copolymer micellar system is technically challenging to achieve.An interesting approach for optimizing the properties and overcoming some of the disadvantages of the single micelles is the combination of two or more distinct amphiphilic polymers in order to assemble the mixed micelles (MMs). 15In comparison to the single micelles (SMs), the mixed micellar systems exhibit multiple advantages, including the improvements in the colloidal stabilities, 16 more accurate size control, 17 and the easier ways to incorporate different modications. 18aclitaxel (PTX) conjugated with polyethylene glycol (PEG) to form a prodrug conjugate (PTX-PEG) has been well-established by some previous reports, 10,16,19,20 in which PTX-PEG can selfassemble into the nanoscaled particles in an aqueous solution.However, the signal PTX-PEG micelles could not fully afford the sophisticated requirements for cancer therapy, such as an increased stability and tumor targeting. 21,22In order to construct a promising system that is suitable for cancer therapy, DSPE-PEG-FA was introduced to form the MMs with PTX-PEG.The as-prepared MMs showed a decreased critical micelle concentration as well as specic tumor targeting.The following advantages were expected to be translated in altered pharmacokinetics: longer mean residence time (MRT) of the drug in the bloodstream, increased bioavailability, reduced the administered dose, and possible diminished non-specic organ toxicity as a result of more precise drug delivery to the target tissues.The ability to engineer the mixed micelle based on PTX-PEG could open a new window to reveal the new potential of PTX-PEG and signicantly boost the utility of this already promising polymer for a variety of applications.

Cell culture
Human cervical carcinoma cell line (Hela) was cultured in a folate (FA) free DMEM medium containing 10% fetal bovine serum (FBS, Hyclone, USA), 100 U mL À1 of penicillin, and 100 mg mL À1 of streptomycin at 37 C using a humidied 5% CO 2 incubator (311, Thermo Scientic, USA). of the H22 cells (murine liver cancer carcinoma cells, a gi from Dr Pengfei Cui in China Pharmaceutical University) were incubated subcutaneously to the ank region of the mice.Tumor volume (V) was determined by measuring length (L) and width (W) and calculated by the formula:

Synthesis and characterization of PTX-PEG
The carboxylated PEG was rst synthesized according to the previous report with some modications. 16Briey, succinic anhydride (0.5 mmol, 50 mg) and PEG (0.25 mmol, 500 mg) were dissolved in 2 mL of anhydrous dimethylformamide (DMF) in a round bottom ask.Subsequently, DMAP (0.5 mmol, 61 mg) and triethylamine (TEA, 0.5 mmol, 70 mL) were homogeneously added to the solution under stirring. 16The reaction was carried out at 35 C with N 2 protection for 24 h.At the end of the reaction, iced ethyl ether was added and carboxylated PEG was precipitated.The white product was obtained by ltering ethyl ether, followed by vacuum desiccation for 24 h.
The PTX-PEG was synthesized by conjugation of PTX and carboxylated PEG.Briey, carboxylated PEG (0.1 mmol, $220 mg), NHS (0.8 mmol, 92.1 mg), and DCC (0.2 mmol, 38.3 mg) were dissolved in 5 mL of anhydrous dichloromethane (DCM).The reaction proceeded under the protection of N 2 at room temperature for 24 h to activate the carboxyl of carboxylated PEG.In addition, PTX (0.2 mmol, 170.8 mg) was dissolved in anhydrous DCM in another round bottom ask.The activated PEG uid was added dropwise to the PTX solution under stirring.Subsequently, DMAP (0.2 mmol, 24.4 mg) was added to the mixture.The reaction proceeded under the protection of N 2 at room temperature for 48 h.The mixture was diluted to 40 mL (with DCM) and washed repeatedly with 1% HCl (40 mL) and water (40 mL).The DCM layer, obtained by a separating funnel, was further dried over anhydrous Na 2 SO 4 and concentrated to approximately 1 mL under vacuum at 45 C. Subsequently, iced ethyl ether was added to precipitate PTX-PEG.The white product was obtained by ltering ethyl ether followed by vacuum desiccation for 24 h.
The 1 H NMR spectra of PTX-PEG were recorded using a nuclear magnetic resonance spectrometer (Avance™ 600, Bruker, Germany) that was operated at 300 MHz using CDCl 3 as a solvent.
The mean molecular weights (MMW) of the copolymers were determined using gel permeation chromatography (GPC).The separation of the polymer was carried out on a PLgel MIXED-C column (pore size: 5 mm; dimensions: 7.5 mm Â 300 mm) with DMSO as an eluent at the ow rate of 0.6 mL min À1 .The standard curve was calibrated with narrow dextran monodisperse standards using a differential refractive index detector.

Preparation of micelles
Initially, 10.0 mg PTX-PEG with or without 2.0 mg DSPE-PEG-FA were dissolved in 2 mL dimethyl sulfoxide (DMSO).The mixture was stirred for 2 h at room temperature to ensure the complete solubilization of the polymers.Subsequently, 1 mL of distilled water was added to the polymer solution under stirring with a rate of 5 s a drop.The resultant solution was then placed into a dialysis bag (MWCO 1000 Da, 2L Â 6) and dialyzed against distilled water for 2 days to remove the organic solvents.The nal solution in the dialysis bag was ultrasonicated for 30 minutes in an ice bath by a probe-type ultrasonicator (JY92-2D, Ningbo Scientz Biotechnology Co., Ltd, Nanjing, People's Republic of China), followed by ltration through a 0.45 mm pore-sized microporous membrane and lyophilization.The resultant white powder was stored at À20 C until further use.
The critical micelle concentration (CMC) of SMs and MMs was determined by uorescence spectroscopy (F-2500, Hitachi Co., Japan) similar to the previous report. 23

Particle size and morphology analysis
The particle size and polydispersity index were assessed by dynamic light scattering (DLS) method using Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) at 25.0 C AE 0.1 C at 90 .The morphologies and size distributions were observed by transmission electron microscopy (TEM, Hitachi 7700, Japan) at an accelerating voltage of 80 kV.

Drug loading content
The drug loading contents (DLs) of PTX in the MMs were estimated by ultraviolet (UV) spectrophotometer (TU-1810, Purkinje, China).The maximum absorbance peak of PTX was determined at 227 nm.Briey, the micelle solution was diluted to a constant volume.A specic amount of acetonitrile was added to the solution to destroy the micelles.The absorbance of the solution at 227 nm was measured. 24In addition, a standard curve of PTX was established to determine the PTX content in the micelles.

Stability and hemolysis assay
For the colloidal stability test, the freshly prepared MMs were diluted with a phosphate buffer (PBS, pH 7.4) and 50% FBS at the volume ratio of 1 : 10.The change in particle size was recorded at the predetermined time intervals.For the hemolysis assay, the red blood cells (RBCs) were rst obtained from New Zealand rabbits and diluted to 2% suspension with a saline solution.SMs and MMs were added to the 2% RBCs suspension with the same volume to achieve the designated concentrations (0.1, 0.25, 0.5, 0.75, and 1 mg mL À1 ) and incubated at 37 C for 1 h.In addition, the RBCs suspension was also incubated with saline and distilled water under the same condition as the negative (0% hemolysis) and positive controls (100% hemolysis), respectively.Following this, all the samples were centrifuged at 3000 rpm for 10 min and the absorption values of the same volume of the supernatants, which represented the counts of released hemoglobin, were measured by UV spectrophotometer.

In vitro cellular uptake
Fluorescent probe C6 was encapsulated into the nanoparticles aer the formation of the micelles.Briey, C6 (0.1 mg) in 0.5 mL of DMSO was added dropwise to the SMs or MMs solution (PBS, pH 7.4) under stirring at 150 rpm at room temperature.Subsequently, the nal solution was ultrasonicated for 30 min in an ice bath.The resultant solution was dialyzed against an excess amount of PBS for 12 h using a dialysis bag (MWCO: 1000 Da, 2L Â 4).The nal solution was ltered through a 0.45 mm pore-sized microporous membrane to remove the large particles (if any), lyophilized, and stored at À20 C until further use.To evaluate the cellular uptake of the SMs and MMs in Hela cell line, the cells were seeded in 24-well plates at a density of 1 Â 10 5 cells per well and incubated for 12 h at 37 C to allow their attachment.To investigate whether the nanoparticles were taken up through FA receptor-mediated endocytosis, the cells were incubated with 10 mg L À1 of free FA for 1 h prior to the addition of the nanoparticles.The medium was replaced by a serum-free cell culture medium containing 0.25 mg mL À1 of the C6 loaded SMs or MMs.Aer 2 and 4 h of incubation, the culture media were removed and the cells were rinsed with PBS thrice.Subsequently, the cells were xed with 4% formaldehyde (10 min) and observed under an inverted uorescence microscope (Axio Observer A1, Zeiss, Germany).The quantitative measurement of the C6 uorescence intensity in the cells at each time point was conducted using ow cytometry (FCM, BD FACS Calibur, USA).Briey, at the designated time intervals, the culture medium was removed and all cell samples were washed with PBS for three times.The cells were harvested by trypsinization and collected by centrifugation.Following supernatant aspiration, the cells were washed with PBS twice and then resuspended in 0.5 mL of PBS prior to being subjected to the FCM analysis.

In vivo tumor targetability imaging
When the tumor volume reached about 300 mm 3 , the xenogra H22 tumor xenogra mice were randomly divided into two groups (n ¼ 3) and intravenously administered with the DiR loaded SMs and MMs via the tail vein.At the prearranged time interval aer injection (12 h), the NIR uorescent images were captured by Maestro in vivo Imaging System (CRI Inc., USA).Aer in vivo imaging, the mice were sacriced and their main organs (hearts, livers, spleens, lungs, and kidneys) as well as tumors were excised to measure their individual uorescence intensities.

In vitro cytotoxicity studies
The Hela cells were used to determine the comparative cytotoxicity of a commercial preparation of PTX (Taxol), SMs, and MMs by the MTT assay as reported previously. 25The cells were rst seeded into 96-well plates at the density of 3.0 Â 10 3 cells per well.Aer 12 h of incubation, the primary culture medium was discarded, following with the addition of a fresh serum-free medium containing different formulations at various PTX concentrations.Aer 48 h of incubation, the medium was replaced with an equal volume of the fresh medium containing 20 mL of 5 mg mL À1 MTT and incubated for 4 h at 37 C.Then, MTT was removed and the cells were lysed with dimethyl sulfoxide (DMSO) under stirring for 15 min on a microtiter plate shaker.The viability of the cells was estimated according to the optical density (OD) values determined by a microplate reader at an absorption wavelength of 570 nm (Bio-Rad, model 680, USA).

In vivo antitumor activity
The H22 murine liver cells overexpressing FA receptor 26 was employed to test the in vivo antitumor activity of the nanoparticles.When the tumor volume of the H22 tumor-bearing BALB/c mice reached approximately 100 mm 3 , the mice were randomly divided into four groups (n ¼ 3 for each group) as follows: (1) saline, (2) Taxol, (3) SMs, and (4) MMs.All groups were injected intravenously via the tail vein (noted as day 0).The therapy was repeated 7 times every 2 days.Tumor volume and body weight of each mouse were measured before the injection.At 2 days aer the nal injections, two mice in each group were randomly chosen and sacriced to prepare the tumor sections.The tumors were extirpated, washed thrice with saline, weighed, and then xed in 10% formalin.The formalin-xed tumors were embedded in the paraffin blocks to prepare the hematoxylin and eosin (HE)-stained tumor sections.The sections were then visualized under an inverted uorescence microscope.

Characteristics of PTX-PEG
Fig. 1 shows the 1 H NMR spectra of PTX-PEG.The methyl (a) and methylene (b) peaks of PEG at around 3.37 ppm can be observed.According to the literature, 27 among all hydroxyl groups in PTX, the most suitable position for the structural modication is the C-2 0 -OH.Therefore, the activated carboxylated PEG prefers to react with a hydroxyl group linked with C-2 0 of PTX by forming an ester bond, which would lead to a shi of the neighboring double peak of CH-2 0 hydrogen from d 4.79 ppm to d 6.0 ppm.The clearly observed peak at 6.0 ppm (c) indicated the successful connection of succinic anhydride with PTX.In addition, the characteristic peaks of PTX with aromatic protons at 7.0-8.0ppm (d) also conrmed the conjugation of PTX.The presence of all characteristic peaks of PTX and PEG (Fig. 1) indicated the successful synthesis of PTX-PEG.The GPC data (Fig. S1 †) also revealed that compared with PEG (MMW: 2000), PTX-PEG has an increased molecular weight of around 3500, which further conrmed that PTX-PEG was successfully synthesized.

Morphology and size distributions
The critical micelle concentration (CMC) of SMs and MMs was determined by uorescence spectroscopy using pyrene as a hydrophobic probe (Fig. S2 †).The CMC of SMs and MMs are 0.00700 and 0.00139 mg mL À1 , respectively.The low CMC value ensures their stability when being diluted in vivo.The morphology and size of SMs and MMs are shown in Fig. 2. It can be observed from the TEM images that the uniform spherical micelles are formed in the two samples.The size and size distribution of the micelles were measured by DLS.The average size of the SMs is 56.9 nm with the polydispersion index (PDI) of 0.173 (Fig. 2A), while the average size of the MMs is 78.0 nm with the PDI of 0.146 (Fig. 2C), indicating that the introduction of DSPE-PEG-FA resulted in a slightly increased size without changing the micelle-forming ability of PTX-PEG.It is reported that the nanoparticles with a size of 10-200 nm can passively accumulate in the tumor cells via the enhanced penetration and retention effect. 28The graed PEG chain could also increase the hydrophilicity of the micelles and serve as a shielding polymer to avoid the potential recognition by the reticuloendothelial system that is primarily responsible for the capture of ectogenic nanoparticles.Moreover, FA as a widely adopted targeting moiety was capable of specically targeting the FA receptor that overexpressed in various types of cancer.The introduction of FA offers the possibility to active targeting cancers with an enhanced antitumor efficacy that has been employed and conrmed by many previous reports. 29,30Herein, the MMs formed by a rational mixture of DSPE-PEG-FA with PTX-PEG were expected to combine both passive and active targeting abilities to elevate the antitumor efficacy as compared to the SMs.

Drug loading content
We prepared the micelles by a dialysis method.The maximum absorbance peak of PTX was determined at 227 nm (Fig. S3 †).The drug loading content (DLC) of PTX in the SMs and the MMs was calculated by the following formula: DLC (%) ¼ weight in PTX-PEG/weight of PTX-PEG Â 100%, which was 20.3% and 16.4%, respectively.

Stability and hemolysis assay
There are some basic requirements for an ideal drug delivery system intended to safely deliver the encapsulated drug molecules to bypass multiple extracellular barriers and locate specically to the targeted site.It has been generally recognized that the nanoparticles should be able to maintain their morphology in the physiological conditions without a signicant size change for at least a certain period of time, since the View Article Online particle size is a critical parameter to determine the in vivo fate of the drug delivery system.Moreover, the drug delivery system should not induce any adverse reactions, such as hemolysis, during the entire process.As a result, the MMs were studied regarding their stability as well as hemolysis on time and concentration-dependent manner.To estimate the colloidal stability of the described nanoparticles, the change in particle size of the MMs, which serves as an indicator of the potential instability under the physiological conditions, was monitored in PBS (pH 7.4) and 50% FBS.As shown in Fig. 3A and S4, † the size of the MMs remained constant during the entire period with only slight uctuation, which indicated that the asprepared delivery system was capable of maintaining its size without being affected by the environment for a long time.This result offers the potential for controllable drug delivery for cancer therapy and is benecial for its further development as a stable platform with a predictable behavior to t more extensive applications.Apart from the steadily delivering and controlled release properties, hemolysis of the drug-loaded MMs was also investigated as a safety guide for intravenous administration and medicinal application. 31As shown in Fig. 3B, the drug-loaded MMs displayed no or neglectable hemolysis (merely 1.79%) on RBCs at the highest concentration of 1 mg mL À1 .Considering that when applied in vivo, the MMs would be diluted to a much lower concentration compared with those being tested in our study, we therefore suggest that the as-prepared MMs could be relatively safe nanoparticles without inducing noticeable hemolysis.Collectively, we demonstrated that the MMs exerted a stable prole with a high biocompatibility, which holds a great promise to benet the therapeutic effect in vivo.

Cellular uptake
PTX-PEG and DSPE-PEG-FA could automatically form the core-shell structured micelles in an aqueous solution on reaching their CMC.The advantages of this structure offer a preferable protection to the drugs encapsulated in the core, while the shell composed of PEG can shield the nanoparticles from the attack of plasma proteins.Moreover, the FA segment exposed towards the surface of the nanoparticles can target the folate receptors that are excessively expressed in the tumor cells.C6 as a uorescent molecule capable of indicating the location and the endocytosis kinetics of nanoparticles was encapsulated in the micelles. 32The cells with or without FA pretreatment were incubated with the C6-loaded SMs and MMs and observed by uorescence microscopy.The C6 uorescence intensity was further quantied by FCM at different time points.The Hela cells overexpressing FA receptors were used for the cellular uptake evaluation. 33s shown in Fig. 4A, the uorescence microscopy images from both systems revealed the bright green uorescence signals in the cytoplasm of the Hela cells, which was attributed to C6, indicating that the C6-incorporated nanoparticles successfully entered the cells.It could be also observed that an extended incubation time from 2 h to 4 h resulted in more intense uorescence in the cell.This suggested that the cellular uptake of both SMs and MMs was positively related to the  incubation time.However, under the same conditions, the C6loaded MMs displayed a remarkably higher intensity compared with that for the SMs, which was also conrmed by the quantitative analysis by FCM as the uorescence signals in the MMs treated cells was 2.15-fold to that of the SMs treated cells (Fig. 4B).To address the concept of FA receptor-targeting, the cells were pretreated with an excess FA solution and then incubated with the SMs and the MMs for 4 h.The FA untreated cells subjected to the same procedures were employed as a blank control.As displayed in Fig. 4C and D, the FA pretreatment resulted in a signicant decrease in the cellular uptake in the MMs treated cells, but no visible changes were observed in the SMs treated cells.The FCM results revealed that the uorescence signals in the MMs treated Hela cells dropped to 50% of that of the untreated cells.These results strongly suggested that the cellular uptake of the MMs was via the FA receptor-mediated pathways that contributed signicantly to enhance the uptake of the nanoparticles as compared with the SMs.

In vivo imaging
The introduction of DSPE-PEG-FA to the MMs was expected to increase the accumulation of the micelles at the tumor site.
Herein, the uorescence intensity of the DiR loaded MMs was monitored in comparison with the unmodied DiR-loaded SMs using a NIR uorescence imaging system for 12 h to evaluate the targeting ability of different nanoparticles in the H22 tumorbearing mice. 34Fig. 5A shows the in vivo images at the tumor site aer the intravenous injection of the DiR loaded SMs and MMs at different time points.Signicant differences in the targeting efficacy between SMs and MMs are observed.In detail, the uorescence intensity of the MMs at the tumor site is markedly stronger than that of the SMs at 12 h post-injection, indicating the preferential accumulation of the MMs at the tumor site.This conclusion was further veried by the ex vivo imaging of the tumor and major organs (Fig. 5B), demonstrating that the SMs, whose distribution in the tumor tissue was inferior to that of the MMs, have only passive targetability.
The powerful tumor targetability of the MMs could be ascribed to a combination of the EPR effect and the FA-mediated endocytosis mechanism.

In vitro cytotoxicity
The comparative in vitro cytotoxicity of Taxol, SMs, and MMs were investigated in the Hela cells for 24 h and 48 h.As shown in Fig. 6A and B, all three formulations exerted cytotoxicity on the Hela cells to some extent.The cytotoxicity effect was positively related to the PTX concentration and the exposure time.It was observed that the MMs exerted the best cytotoxicity effect with comparable cell viability to the cells aer 24 h of incubation, which is superior to that of the SMs at the same condition.Moreover, aer incubation for 48 h, the MMs showed more potent cytotoxicity than Taxol at all tested concentrations.These results suggested that the MMs could have an enhanced cytotoxicity to the Hela cells, which would be benecial for the in vivo antitumor applications.

In vivo anti-tumor
To conrm the antitumor potential of the MMs in vivo, the antitumor efficacy was evaluated in a subcutaneous H22 tumor xenogra model.As shown in Fig. 7A, the H22 tumor-bearing mice in the saline-treated group showed a rapid increase in the tumor growth over the entire period of the experiment.Moreover, the groups treated with Taxol and SMs exhibited a higher growth delay in comparison with the saline control  group, which conrmed the antitumor effect of PTX.Interestingly, more elevated suppression of the tumor growth during the treatment with the MMs than other PTX-containing groups was observed.Fig. 5B shows the variation in the body weight of the mice with time.The body weight of the mice in the saline and Taxol groups began to steadily decrease aer a period of time aer administration, indicating that the living quality of the mice was compromised by the tumor burden and/or the side effects.In contrast, no noticeable body weight loss was observed in both SMs and MMs groups.In particular, in the MMs treated groups, the body weight of the mice was increased as compared to that at the initial assessment, implying that the tumor-homing property of the MMs not only increases their anti-cancer efficacy, but also reduces their safety risks.The representative HE-stained tumor sections from different experimental groups of the mice are displayed in Fig. 5C.The group treated with saline displayed the typical pathological characteristics of the tumor, including the closely packed tumor cells and the heavily stained cytoplasm.The images from other PTX-containing groups showed a cancer cell remission to some extent, while the group administered with the MMs displayed a massive remission with the most powerful antitumor ability.These observations presented substantial evidence to the stronger antitumor effect of the MMs.Overall, the MMs constructed in this study had a promising potential as a preferable tumor-targeting DDS for an efficient cancer therapy.

Conclusion
In summary, the paclitaxel prodrug mixed micelles (MMs) were successfully developed for tumor-targeted chemotherapy.
The experimental results indicated that drug loading of PTX into the paclitaxel prodrug MMs was as high as 16.4%, which is superior to many currently available carriers.Furthermore, the paclitaxel prodrug MMs showed high biocompatibility with enduring colloidal stability and negligible hemolysis.Moreover, the paclitaxel prodrug MMs increased the accumulation of PTX in the Hela cancer cells via FA receptormediated endocytosis with an enhanced tumor-homing capability in the H22 tumor-bearing mice.In vitro and in vivo anticancer efficacy assays revealed that the MMs possessed a preferable anti-cancer ability, which exhibited the minimized toxic side effects of PTX and a strong tumor-suppression potential in the clinical application.

Fig. 4
Fig. 4 The in vitro cellular uptake analysis of SMs and MMs in the Hela cells.(A) The inverted fluorescence microscope images of the cells incubated with the C6-loaded SMs and MMs for 2 and 4 h.Scale bar: 200 mm.(B) Flow cytometric analysis of mean fluorescence intensity in the cells treated with the C6-loaded SMs and MMs for 2 and 4 h.(C) Inverted fluorescence microscope images of the cells incubated with the C6loaded SMs and MMs with or without the FA pretreatment 4 h.Scale bar: 200 mm.The data are shown as mean AE S.D. (n ¼ 3).(B) Flow cytometric analysis of mean fluorescence intensity in the cells treated with the C6-loaded SMs and MMs with or without the FA pretreatment 4 h.

Fig. 3 (
Fig. 3 (A) Colloidal stability of MMs in PBS (pH 7.4) at 37 C for up to 48 h.(B) Hemolysis of MMs at various concentrations.The data are shown as mean AE S.D. (n ¼ 3).

Fig. 5
Fig. 5 In vivo imaging and biodistribution of the H22 tumor xenograft mice after injection of DiR-loaded SMs and MMs.(A) NIR fluorescence images of nanoparticle localization in the mice (upper) as well as their main organs and tumor (lower) after the intravenous injection of the DiRloaded SMs and MMs for 12 h.(B) Semiquantitative analysis of biodistribution of the DiR-loaded SMs and MMs in the mice determined by MFI in the tumor or tissues.The data are shown as mean AE S.D. (n ¼ 3).

Fig. 6
Fig. 6 Cell viabilities of the Hela cells incubated with Taxol, SMs, and MMs at various PTX concentrations for 24 h (A) and 48 h (B).The data are expressed as mean AE S.D. (n ¼ 3).

Fig. 7
Fig. 7 The tumor volume (A), body weight (B), and HE staining of tumor tissue (C) analysis of the H22 tumor-bearing BALB/c mice after the intravenous injection administration of different formulations (saline, Taxol, SMs, and MMs).The measurement of tumor volumes and the injection of formulations were repeated every 2 days for two weeks.Dose: 10 mg kg À1 PTX per mouse.The data are expressed as mean AE S.D. (n ¼ 3).