Development , Characterization , and Evaluation of PSMA-Targeted Glycol Chitosan Micelles for Prostate Cancer Therapy

Prostate cancer-binding peptides(PCP-)modified polymericmicelles were prepared and used for the treatment of prostate-specific membrane antigen(PSMA-) expressing prostate cancer in a target-specificmanner. Cholesterol-modified glycol chitosan (CHGC) was synthesized. PCP-conjugated CHGC (PCP-CHGC) micelles were fabricated and characterized. The degree of substitution was 5.2 PCP groups and 5.8 cholesterol groups per 100 sugar residues of glycol chitosan. The critical aggregation concentration (CAC) of PCP-CHGC copolymer was 0.0254mg/mL. Doxorubicin (DOX) was chosen as a model antitumor drug. The DOX-loaded micelles were prepared by an o/w method. The mean diameter of DOX-loaded PCP-CHGC (DOX-PCP-CHGC) micelles was 293 nm determined by dynamic light scattering (DLS). DOX released from drug-loaded micelles was in a biphasic manner. DOXPCP-CHGCmicelles exhibited higher cytotoxicity in vitro against PSMA-expressing LNCaP cells than DOX-loaded CHGC (DOXCHGC) micelles. Moreover, the cellular uptake of DOX-PCP-CHGC micelles determined by confocal laser scanning microscopy (CLSM) and flow cytometry was higher than that of DOX-CHGCmicelles in LNCaP cells. Importantly, DOX-PCP-CHGCmicelles demonstrated stronger antitumor efficacy against LNCaP tumor xenograft models than doxorubicin hydrochloride and DOXCHGCmicelles. Taken together, this study provides a potential way in developing PSMA-targeted drug delivery system for prostate cancer therapy.


Introduction
Prostate cancer is one of the most frequently diagnosed cancers among men in the United States and is the second leading cause of male cancer death with 233,000 new cases and an estimated 29,480 deaths in 2014 [1].Current treatment for prostate cancer commonly involves surgery, radiation therapy, hormone administration, and/or chemotherapy [2,3].In the early stage of prostate cancer, the surgical or radiation ablation therapies are relatively effective.Unfortunately, there is no effective therapy for men with metastatic prostate cancer.Clinical data have exhibited that chemotherapeutic agents may prolong survival in a subset of patients.Nevertheless, the dose and duration of administration of these drugs were limited, due to the systemic toxicity and the lack of sufficient selectivity towards tumor cells.Thus, it is an urgent medical need to develop drug delivery systems that selectively transport the antitumor agent into the tumor tissues and cells.Nanosized carriers such as liposomes, micelles, and polymeric nanoparticles have demonstrated improved therapeutic index with minimal side effects [4][5][6].
In recent years, polymeric micelles have received much attention, particularly due to the great potential as drug delivery vehicles in cancer chemotherapy [7][8][9][10].The amphiphilic copolymers can spontaneously form nanostructures with hydrophobic cores and a hydrophilic outer shell in aqueous medium.The hydrophobic core can act as the drug-loading depot, while the surrounding hydrophilic shell can increase stabilization.Moreover, the micelles possess unique properties such as increasing blood circulation time in vivo and passive accumulation in tumor tissue by the enhanced permeability and retention (EPR) effect [11][12][13].In particular, the biodegradable and nontoxic polymeric conjugates, such as glycol chitosan-based amphiphiles, attracted considerable interests [14][15][16].Glycol chitosan self-assembled nanoparticles demonstrated high tumor accumulation that was useful for in vivo delivery of hydrophobic drugs or genes [17][18][19].In our previous study, amphiphilic cholesterol-modified glycol chitosan (CHGC) was synthesized and formed aggregated nanoparticles in aqueous solution [20].Moreover, the DOXloaded CHGC nanoparticles showed the prolonged circulation in blood and exhibited stronger antitumor activity against S180 tumor-bearing mice by the EPR effect than doxorubicin hydrochloride.Further, the nanoparticle surface can be functionalized to attain active targeting, leading to improvement of drug intratumoral accumulation.This can be achieved by tagging target-specific moieties or ligands onto the micelle's surface [21][22][23].Such ligands are recognized by specific receptors which are either uniquely expressed or overexpressed on certain types of cancer cell surfaces.Several targeting moieties, such as folate, aptamer, or peptides, have been successfully conjugated with the carriers to deliver antitumor drugs or genes for treatment of prostate cancer [24][25][26].
One promising candidate for targeted prostate cancer therapy is prostate-specific membrane antigen (PSMA), a 100 kDa membrane-bound glycoprotein, which is highly overexpressed on the surface of human prostate cancer cell lines (such as LNCaP and CWR22R) and tumor-associated neovasculature in a variety of solid tumors [27,28].In prostate cancer tissues, PSMA is expressed at levels > 1000fold greater than that in other tissues, such as brain and proximal small intestines [29].Hence, extensive efforts have been devoted to investigating PSMA receptor binding ligands [3].For example, the peptide phage display approach has been widely used to search for ligands that bind epitopes on the cell surfaces in vitro and in vivo.Romanov et al. performed search for peptide ligands to PSMA-expressing LNCaP cells receptors, and they found that the prostate cancer-binding peptides (DPRATPGS sequence) were the best binder to the surfaces of LNCaP cells [30].Based on the above considerations, we attempted to use PCP as the targeting moiety to fabricate targeted drug delivery system.
Herein, the purpose of this study was to develop PSMAtargeted polymeric micelles for prostate cancer therapy.PCPmodified CHGC conjugate was synthesized and characterized.The physicochemical properties of PCP-CHGC micelles were then investigated.DOX, a typical cytotoxic anthracycline antibiotic, was chosen as antitumor model drug and encapsulated into the PCP-CHGC micelles.Further, in vitro cellular uptake and cytotoxicity of DOX-load micelles were evaluated in prostate cancer cell lines, LNCaP and PC-3.The antitumor efficacy studies of drug-loaded micelles were also investigated in LNCaP-bearing nude mice.
Male BALB/c nude mice (4 weeks of age, Hunan SLAC Jingda Laboratory Animal Co. Ltd., Changsha, China) were maintained in a pathogen-free condition.All animal experiments were performed according to the international regulations for animal experimentation.

Synthesis of CHGC Conjugate.
CHGC conjugate was synthesized by using our previous method with little modification [31].Briefly, cholesterol and succinic anhydride were mixed and reacted for 24 h.Then cholesterol hemisuccinate was obtained by purification and recrystallization.Glycol chitosan (500 mg) was dispersed in 30 mL of distilled water overnight and was diluted with 106 mL of ethanol under quick stirring.EDC (58.3 mg) and cholesterol hemisuccinate (74.0 mg) were dropwise added.After 72 h, the mixture was sequentially dialyzed (MWCO: 3.5 kDa) against the excess amount of ethanol/distilled water solution (88 : 12, v/v) and distilled water.Finally, the dialyzed solution was filtered through 0.8 m membrane and lyophilized to obtain CHGC copolymer.

Synthesis of PCP-CHGC Conjugate
. CHGC (100 mg) was dissolved in 40 mL of distilled water (pH 8.0) and followed by sonication using a probe type sonifier (Ningbo Scientz Biotechnology Co. Ltd., Ningbo, China) at 200 W for 40 times in an ice bath.Then the coupling agent SMCC (10.8 mg) was added, which was dissolved in 0.5 mL of DMSO.The solution was stirred at room temperature for 2 h.After adjusting the pH value of the mixture to 6.8, PCP (Ac-CDPRATPGS sequence, 45.2 mg) were introduced.After further 4-hour reaction, the unconjugated PCP were discarded by using the centrifugal-ultrafiltration method [33].The resulting solution was dialyzed (MWCO: 3.5 kDa) against distilled water for 24 h.The purified dialysate was freeze-dried and stored at −20 ∘ C.

Preparation of DOX-Loaded Micelles.
The DOX-loaded micelles were prepared by the emulsion/solvent evaporation method [34,35].The blank copolymer micelles were prepared by probe sonication as stated above.DOX base was produced by reaction of DOX⋅HCl with 3-mole equivalent of triethylamine in chloroform overnight.3 mL of DOX (4.5 mg) solution was added to 60 mL of blank micelles (30 mg, CHGC or PCP-CHGC) under vigorous stirring to form the o/w emulsion.Then the mixture solution was placed into a flask and evaporated under reduced pressure condition, allowing removal of chloroform and formation of self-assembled micelles.The aqueous suspension was filtered through a 0.8 m membrane and eluted through a Sephadex G25 fine column to remove nonencapsulated DOX.The DOX-loaded micelles were lyophilized and kept at −20 ∘ C for future use.

2.5.
Characterization. 1 H-nuclear magnetic resonance (NMR) spectra were recorded on Avance DMX500 spectrometer using D 2 O as the solvent.Chemical shifts are shown in parts per million (ppm).
The cholesterol substitution degree in the conjugates was investigated by a colloidal titration method, which is based on the reaction between positively charged glycol chitosan and negatively charged potassium poly(vinyl sulfate) [15,36].Briefly, 5 mg of CHGC was dissolved in 2% acetic acid solution (5 mL) overnight.Then, 100 L of the indicator and 0.1% (w/v) toluidine blue were added under a stirring condition.The CHGC suspension was slowly titrated with 2.5 mM potassium poly(vinyl sulfate) solution until the opaque solution suddenly became clear.Another 5 mL of distilled water was used as blank assay.
The substitution degree of PCP in the PCP-CHGC conjugate was measured by Ellman's assay, which can detect free sulfhydryl groups in solution.After the synthesis reaction, the unconjugated prostate cancer-binding peptides were removed by the centrifugal-ultrafiltration method.Thereafter, the unreacted PCP or initially fed PCP was dissolved in Ellman's working solution.Ellman's reagent solution was immediately added and stirred for 15 min at room temperature.After filtration, the solution was measured by ultraviolet spectrometer (UV-1206, Shimadzu Corp., Japan) with the absorbance at 412 nm.The amounts of unattached PCP and initially fed PCP were determined.Then the amount of PCP groups conjugated to PCP-CHGC was calculated by the subtraction method.
The critical aggregation concentration (CAC) of micelles was investigated by using pyrene as a florescent probe.The PCP-CHGC conjugate suspension was prepared by sonication as described above and adjusted to various conjugate concentrations.A known amount of pyrene in acetone was added into each of the 10 mL vials, and acetone was evaporated.A total of 10 mL of various concentrations of PCP-CHGC conjugate were introduced into each vial and heated at 50 ∘ C for 10 h to equilibrate pyrene and the micelles.And the mixture solution remained undisturbed to cool overnight at room temperature.The final concentration of pyrene was 6.0 × 10 −7 M. Steady-state fluorescent spectra were measured using a fluorescence spectrophotometer (Hitachi F-4500, Japan).The excitation and emission wavelengths were set at 339 and 390 nm, respectively.The slit width was 2.5 nm.
The hydrodynamic diameters of the micelles were determined by dynamic light scattering (90Plus, Brookhaven Instruments Corp., Holtsville, NY, USA).Transmission electron microscopy (TEM, JEM-1230, Japan) was employed to observe the morphology of the micelles at an acceleration voltage of 80 kV.TEM sample was prepared by placing a drop of micelles onto a 300-mesh copper grid coated with carbon.The extra solution was blotted with filter paper, followed by air drying.
The DOX-loading content (LC) and entrapment efficiency (EE) of DOX-loaded micelles were investigated by using a UV-Vis spectrophotometer (UV-1206, Shimadzu Corp., Japan).The freeze-dried samples were dispersed in aqueous solution, and DMSO was added to dissociate the micelles.The absorbance at 481 nm was performed to detect the DOX concentration.The LC and EE were calculated by LNCaP cells were seeded at a density of 1 × 10 6 cells/well in 6-well plates.After the cells were allowed to adhere for 48 h, the medium was replaced with DOX⋅HCl, DOX-CHGC, or DOX-PCP-CHGC micelles (equivalent DOX concentration: 10 g/mL) in RPMI 1640 medium supplemented with 10% FBS.After 4 h treatment, the cells were rinsed with cold PBS (pH 7.4), subsequently detached by trypsinization.Then, the cells were resuspended with PBS before the DOX fluorescence intensity was determined using a flow cytometer (Becton Dickinson FACSCalibur, USA) equipped with 488 nm argon ion laser.The data were the mean fluorescent signals for 10,000 cells.Each experiment was conducted in three times.
2.9.In Vitro Cytotoxicity.The in vitro cytotoxicity of free DOX, blank micelles, and DOX-loaded micelles was investigated by MTT assay in PSMA-positive LNCaP and PSMAnegative PC-3 cells.Briefly, the cells were plated in 96-well plates at a density of 1 × 10 5 cells/well and cultured to allow them to adhere at the bottom of the plates.Then, different concentrations of blank micelles, DOX⋅HCl, or DOX-loaded micelles were added and further incubated for 24 h.After that, the medium in each well was discarded.200 L of fresh medium and 30 L of MTT solution with a concentration of 5 mg/mL were added and incubated for further 4 h at 37 ∘ C. Thereafter, the solution was removed and 100 L of DMSO was added to dissolve MTT formazan crystals.After 15 min incubation, the absorbance of formazan product was measured at 570 nm in a microplate reader (Thermo Scientific Multiskan MK3, Hudson, USA).All the experiments were performed in triplicate.On the 20th day of the therapy, mice were euthanized and the tumors were collected.Then the organs were fixed with paraformaldehyde for 48 h and embedded in paraffin.The sections were stained with hematoxylin and eosin (H&E) to assess the antitumor activity of different treatments via optical microscope observation (Nikon 80i, Japan).

In Vivo Tumor Inhibition
2.11.Statistical Analysis.Data were described using the mean ± standard deviation (SD).The statistical analysis was performed by a one-way ANOVA with a Bonferroni post hoc test.The level of significance was set as  < 0.05.

Synthesis and Characterization of PCP-CHGC.
The chemical structure of PCP-CHGC was shown in Figure 1.Before the PCP modification, the CHGC was synthesized by the reaction between carboxyl group of cholesterol hemisuccinate and amino group of glycol chitosan using EDC as the coupling reagent.And the chemical structure of CHGC was confirmed by 1 H NMR spectra (Figure 2).Compared with glycol chitosan, the new signal at 1.27 ppm belonged to methene hydrogen of cholesterol hemisuccinate group, indicating that CHGC conjugate was successfully synthesized [31,32].According to the colloidal titration test, the degree of substitution was 5.8 cholesterol groups per 100 sugar residues of glycol chitosan.In addition, PCP groups were conjugated   to the distal end of CHGC in the presence of SMCC, which is a heterobifunctional cross-linking reagent incorporating an extended spacer with amine and sulfhydryl reactivity.Compared with the 1 H NMR spectrum of CHGC, PCP-CHGC exhibited principal peaks related to the PCP moieties (4.16, 3.23, 3.10, 1.55, 1.16, and 0.87 ppm).These results revealed that PCP-CHGC was successfully synthesized.Moreover, Ellman's assay was used to determine the amount of PCP group conjugated on the glycol chitosan backbone.The degree of substitution was 5.2 PCP groups per 100 sugar residues of glycol chitosan.

Characterization of Blank Micelles.
The synthesized PCP-CHGC was able to self-assemble to form micelles in aqueous  solution.As shown in Table 1, the mean diameters of CHGC and PCP-CHGC micelles analyzed by DLS were 228 and 246 nm, respectively.This result indicated that particle size of PCP-CHGC micelles increased after the modification of PCP groups on the outer shell of micelles.
The CAC of PCP-CHGC copolymer was detected by fluorescence spectrophotometry with pyrene as a fluorescent probe.As shown in Figure 3, the value of I 338 /I 333 ratio was changed with increasing concentrations of the conjugate.It indicated that pyrene molecules were transferred from a water environment to a hydrophobic micellar microdomain [37].The CAC of PCP-CHGC measured by fluorescence spectrometry was 0.0254 mg/mL, which suggested that PCP-CHGC micelles can form nanosized nanoparticles under highly diluted condition.

Preparation and Characterization of Drug-Loaded
Micelles.The physical entrapment of DOX into polymeric micelles was achieved by an emulsion/solvent evaporation method.Triethylamine was used to remove hydrochloride from DOX⋅HCl [38].In aqueous medium, DOX-PCP-CHGC micelles can be formed.CHGC and PCP-CHGC micelles showed good loading capacities for DOX (Table 1).The loading content of DOX-CHGC and DOX-PCP-CHGC micelles was 10.5% and 11.4%, respectively.This result indicated that the modification of PCP in PCP-CHGC micelles showed no significant effect on the drug loading into micelles ( > 0.05).Additionally, the encapsulation efficiency of DOX-CHGC and DOX-PCP-CHGC micelles was >80%, suggesting the good loading behavior of these polymeric micelles.
The particle size of DOX-loaded micelles was shown in Table 1.The mean diameters of DOX-CHGC and DOX-PCP-CHGC micelles were 274 and 293 nm, respectively.We noted that the size of DOX-loaded micelles was larger than that of drug-free micelle counterparts.This result was ascribed to the fact that DOX molecules were loaded into the micellar inner space.As shown in Figure 4, it is evident that the DOX-PCP-CHGC micelles are in a regular spherical shape.The mean diameter of DOX-PCP-CHGC micelles determined by TEM was about 240 nm, which was smaller than hydrodynamic size analyzed by DLS.This is due to the solvent effect.The particles size observed by TEM is the diameter of air-dried micelles, whereas the size measured by DLS is the hydrodynamic diameter of micelles in the hydrated state.Similar phenomena were also described in other literatures [39,40].

In Vitro Drug
Release.As shown in Figure 5, DOX from DOX-loaded micelles in vitro was investigated in PBS (pH 7.4).The cumulative DOX release from DOX-CHGC and DOX-PCP-CHGC micelles over 72 h was 50.4% and 49.1%, respectively.It was seen that DOX-CHGC and DOX-PCP-CHGC micelles exhibited similar release behavior.The PCP conjugation did not have significant effect on the release behavior of DOX-PCP-CHGC micelles.Moreover, DOX released was in a biphasic way, which showed an initial fast release at the first 6 h and was followed by a step of sustained release for up to 72 h.As previously described, the initial moderately fast release and sustained release drug system may offer the advantage of rapid drug effect and prolonged drug activity on the tumor [41].Additionally, this release kinetic is related to both dissolution and diffusion based on the Noyes-Whitney law of dissolution and Fick's first law of diffusion [42].

In Vitro Cellular Uptake Study.
The cellular uptake and distribution of micelles were monitored under CLSM and flow cytometry.As shown in Figure 6, the red fluorescence originated from DOX formulations.After incubating PSMApositive LNCaP cells with DOX-CHGC or DOX-PCP-CHGC micelles for 4 h, the weak DOX fluorescence in cytoplasm was observed.In addition, the majority of visible red fluorescence was found in nuclei.It can be ascribed to the released DOX from the micelles.These results indicated that the micelles were taken by LNCaP cells through endocytosis.Moreover, the red fluorescence from the internalized DOX-PCP-CHGC was apparently brighter than that from DOX-CHGC, indicating that the cellular uptake for DOX-PCP-CHGC micelles was enhanced by PCP modification at the surface of micelles.It should also be noted that stronger DOX fluorescence was observed in the cells following incubation with free DOX⋅HCl, compared with DOX-CHGC and DOX-PCP-CHGC micelles.This result was due to the different internalization mechanisms of the DOX-loaded micelles and DOX⋅HCl.The DOX-loaded micelles were internalized through an endocytosis pathway, whereas DOX⋅HCl molecules were transported into the cells in a passive diffusion manner [43,44].DOX molecules from the micelles diffused into the cells more slowly than DOX⋅HCl in solution without micelle encapsulation.
To further determine the cellular uptake of DOX⋅HCl, DOX-CHGC, and DOX-PCP-CHGC micelles by LNCaP cells after 4 h incubation, flow cytometry was applied.As shown in Figure 7  than the cells treated with DOX-CHGC micelles.These results were in good agreement with those of CLSM experiments.Therefore, the PCP-conjugated micelles exhibited enhanced cellular uptake in LNCaP cells in vitro compared to nonconjugated micelles.This result indicated that PCPmodified micelles could facilitate and improve cellular uptake of micelles in tumor tissues.
3.6.In Vitro Cytotoxicity.In vitro cytotoxic activity of DOX-loaded micelles against LNCaP (PSMA+) and PC-3 (PSMA−) cells was evaluated by using MTT assay. of DOX-loaded CS-SA micelles against C6 cells was less potent than that of DOX⋅HCl [45].This is due to the fact that DOX⋅HCl could be quickly delivered into cells by passive diffusion and instantly inhibit cell growth under the in vitro condition.Moreover, we can see that the cytotoxicity of DOX-PCP-CHGC is lower than that of DOX-CHGC.This result demonstrated that DOX-PCP-CHGC with PCP modifications could not perform the targeting capability in PSMA-negative PC-3 cells.As shown in Figure 8(b), the IC 50 values of blank CHGC and PCP-CHGC micelles were 870.1 and 983.1 g/mL, respectively.These results revealed that these blank micelles showed low cytotoxicity against the test cells.
The cytotoxicity of three DOX formulations against LNCaP cells for 24 h was shown in Figure 9(a).The IC 50 of DOX⋅HCl was 1.37 g/mL, while that of DOX-CHGC and DOX-PCP-CHGC micelles at the equivalent DOX concentration was 6.82 and 3.48 g/mL, respectively.DOX-loaded micelles revealed lower cell killing efficiency as compared to DOX⋅HCl.DOX-PCP-CHGC micelles showed significantly greater cell killing potency against LNCaP cells in contrast with DOX-CHGC micelles ( < 0.05).The decoration of PCP molecules on the micelles could help to enhance their recognition to LNCaP tumor cells and to facilitate intracellular drug delivery through the PSMA-mediated endocytosis, leading to the increase of cytotoxicity of DOX-PCP-CHGC micelles.These results agreed well with the cellular uptake study.Notably, it was unlikely that such a high concentration of DOX⋅HCl would be present in the tumor site during the blood circulation in vivo.And DOX-loaded micelles may facilitate its accumulation at tumor tissue by the EPR effect as previously described [31,43].For comparison, the cytotoxicity of the empty micelles was also investigated.As shown in Figure 9(b), the viability of empty CHGC and PCP-CHGC micelles against LNCaP cells after 24-hour treatment was nearly 70% even at high concentration of 1 mg/mL.This result exhibited that empty micelles had good biocompatibility.Therefore, we inferred that PCP-modified micelles could be a potential carrier for PSMA-mediated targeting drug delivery.And DOX⋅HCl group exhibited the strongest cellular uptake in vitro.As previously reported, free DOX showed rapid clearance from rat plasma [20].However, DOX encapsulated in the polymeric nanoparticles exhibited delayed blood clearance.The long-circulating characteristic of the polymeric nanoparticles endows EPR effect more valuable in tumor targeting [11].Therefore, the EPR effect in solid tumors could contribute to the enhanced antitumor activity of DOX-loaded polymeric micelles.Furthermore, improved tumor targeting of in vivo DOX delivery was seen in the PCP-modified micelles when compared to that of nontargeted micelles.These results indicated that administration of DOX-PCP-CHGC could accumulate in the same tumor tissues with PSMA-mediated pattern.

In Vivo Tumor Inhibition
At the end of the experiment, the images of isolated tumors for different treated groups confirmed the superior efficiency of DOX-PCP-CHGC micelles (Figure 10 To further assess the therapeutic efficacy, histological analysis of tumor sections was performed on the 20th day after the first injection.From the H&E staining in Figure 11, we can find that, in comparison with 5% glucose, blank micelle, DOX⋅HCl and DOX-CHGC groups, DOX-PCP-CHGC group exhibited the greatest massive cancer cell remission and distinct apoptosis degrees.Therefore, DOX-PCP-CHGC micelles could provide better antitumor efficiency with low side effects by enhancing accumulation in tumors and improve cellular uptake.

Conclusion
In the present work, novel PCP-CHGC copolymer was successfully synthesized and characterized.DOX was physically encapsulated into PCP-CHGC micelles with high drugloading capacity.In vitro cytotoxicity and cellular uptake studies revealed that the PCP-modified micelles could specifically recognize the PSMA-positive LNCaP cells and enhance intracellular DOX delivery by PSMA-mediated endocytosis.Furthermore, DOX-PCP-CHGC micelles demonstrated better tumor inhibition in LNCaP-bearing nude mice than DOX⋅HCl and DOX-CHGC micelles.These findings suggested that this formulation design provided promising targeted therapeutics for PSMA-expressing prostate cancer.

Figure 3 :
Figure 3: Plot of the intensity ratio I 338 /I 333 (from pyrene excitation spectra of PCP-CHGC conjugate) as a function of log C.
(b)).Meanwhile, the body weight of mice was monitored to evaluate the systemic toxicity of the formulations during the experiment.As shown in Figure 10(c), the treated mice except DOX⋅HCl group exhibited a slight weight change.This might be due to the bearing of tumor.Compared with DOX-loaded micelles groups, an apparent weight decrease was observed in DOX⋅HCl group.It inferred that DOX⋅HCl could not specifically deliver into the tumor cells, and the drug produced the side effects.

Table 1 :
Physicochemical properties of drug-free and DOX-loaded micelles.
a The ratio of DOX to carrier, based on feed amount (mg/mg).b Measured by dynamic light scattering.c Polydispersity index.d Loading content.e Encapsulation efficiency.The results represent the mean ± SD ( = 3).log C (mg/mL)