Targeted sustained delivery of antineoplastic agent with multicomponent polylactide stereocomplex micelle

    Targeted sustained delivery of antineoplastic agent with multicomponent polylactide stereocomplex micelle Kexin Shen, Di Li, Jingjing Guan, Jianxun Ding, Zhongtang Wang, Jingkai Gu, Tongjun Liu, Xuesi Chen PII: DOI: Reference:

S1549-9634(16)30239-8 doi: 10.1016/j.nano.2016.12.022 NANO 1502

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

12 October 2016 4 December 2016 26 December 2016

Please cite this article as: Shen Kexin, Li Di, Guan Jingjing, Ding Jianxun, Wang Zhongtang, Gu Jingkai, Liu Tongjun, Chen Xuesi, Targeted sustained delivery of antineoplastic agent with multicomponent polylactide stereocomplex micelle, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2016.12.022

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ACCEPTED MANUSCRIPT Targeted sustained delivery of antineoplastic agent with multicomponent

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polylactide stereocomplex micelle

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Kexin Shen, MDa,b, Di Li, PhDb, Jingjing Guan, MPHa, Jianxun Ding, PhDb,*,

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Zhongtang Wang, MDc,**, Jingkai Gu, PhDa, Tongjun Liu, MDa,***, Xuesi Chen, PhDb

a

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Jilin University, Changchun 130012, P. R. China

b

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun 130022, P. R. China Department of Radiation Oncology, Shandong Cancer Hospital Affiliated to

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c

Shandong University, Shandong Academy of Medical Sciences, Jinan 250117, P. R.

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China

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*Corresponding author.

**Corresponding author. ***Corresponding author. E-mail addresses: [email protected] (J. Ding), [email protected] (Z. Wang), [email protected] (J. Liu).

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The authors have declared that no competing interest exists.

This research was financially supported by the National Natural Science

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Foundation of China (Nos. 51673190, 51303174, 51603204, 51673187, 51473165,

Word count for abstract: 133. Word count for manuscript: 3702.

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Number of references: 45.

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Number of figures: 6. Number of tables: 0.

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and 81430087).

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Number of Supplementary online-only files: 1.

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ACCEPTED MANUSCRIPT Abstract A c(RGDfC)-decorated polylactide stereocomplex micelle (cRGD-SCM) was

glycol)-block-poly(D-lactide)

poly(ethylene

(4-arm

glycol)-block-poly(L-lactide)

PEG-b-PDLA),

methoxy

(mPEG-b-PLLA),

and

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poly(ethylene

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prepared through the stereocomplex and hydrophobic interactions among 4-arm

c(RGDfC)-poly(ethylene

glycol)-block-poly(L-lactide)

(cRGD-PEG-b-PLLA)

for

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targeted treatment of αvβ3 integrin-positive C26 colon cancer. Doxorubicin (DOX), a model antitumor drug, was loaded into cRGD-SCM with a diameter of approximately 100 nm, and the drug loading efficiency was 45.9 wt.%. cRGD-SCM/DOX with a

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sustained release pattern exhibited prolonged circulation time, upregulated

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accumulation in tumor, enhanced tumor inhibition, and decreased side effects compared with free DOX and non-targeting SCM/DOX in vivo. More interestingly, the

according

to

the

different

types

of

malignancies.

Therefore,

the

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groups

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targeting ligand in the terminal of PEG can be easily replaced with other targeting

cRGD-decorated platform might be a promising targeted drug delivery system for personal chemotherapy clinically.

Key words: Polylactide; Stereocomplex interaction; Multicomponent micelle; Targetability; Chemotherapy

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ACCEPTED MANUSCRIPT Background Cancers with high morbidity and mortality are one of the most common malignant

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diseases worldwide.[1] Although the technologies of diagnosis and treatment of cancers have been greatly developed in recent years, many patients are already in

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advanced stage when diagnosed.[2] Currently, chemotherapy other than excision by surgery remains the most effective therapeutic regimen for various cancers,

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especially for the advanced cancers that are unsuitable for surgical therapy.[3] In addition, chemotherapy is needed by a plenty of patients after the excision of primary tumors.[4] However, the severe side effects and complications of chemotherapeutic

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agents, such as visceral injury and marrow suppression, restrict their application

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prospects.[5] Therefore, it has been the research focus of cancer chemotherapy around the world that how to improve the antitumor efficacy meanwhile decrease the

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serious side effects and complications.[6,7]

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The development of nanotechnology brings great opportunities for emerging chemotherapy formulations with improved properties.[8,9] With the rapid development of nanomedicines, the polymer nanocarriers of antitumor agents have been widely investigated in recent years.[10,11] There are many advantages of polymer nanoparticle-based drug delivery systems, including improved aqueous solubility of free drugs, increased stability of antitumor agents through specially designed formations, prolonged circulation duration of antitumor agents in vivo, upregulated accumulation in tumor tissue through the enhanced permeability and retention (EPR) effect, enhanced endocytosis, and sustained or burst on-demand drug release 4

ACCEPTED MANUSCRIPT depending on the smart design.[7] Benefited from the aforementioned merits, various polymer nanocarriers, such as micelles,[12–15] vesicles,[16,17] nanogels,[18,19] and

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prodrug nanoparticles,[20–23] have been developed vigorously, and some of them have been applied in clinical trials and even in clinic.

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Across all of the nanoscale polymer drug delivery systems, micelles have attracted a great deal of attention due to the spontaneous assembly characteristics,

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modifiable surface, and facile drug loading and controlled release.[24–26] The targeted performances of polymer micelles with various targeting ligands can upregulate the distribution of antitumor drugs in tumor tissues mediated by the specific

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ligand-receptor recognition.[13,27] The highly selective accumulation in lesion sites

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enhances the efficacy, and reduces the side effects and complications of antitumor agents. Although the targeting micellar platforms exhibit great potential in directional

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drug delivery, the difficult adjustments of physicochemical properties and targeting

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ligands limit their wide applications for the multitude of different types of cancers. The emerging multicomponent polymer micelles provided a powerful strategy to conveniently change the characteristics to meet the demands of different cancer chemotherapies.[28–30] However, the insufficient stability of multicomponent micellar systems limits the potential preclinical and clinical applications. In the present study, a stereocomplex-reinforced multicomponent polylactide (PLA) micelle with cyclic(Arg-Gly-Asp-D-Phe-Cys) (c(RGDfC), cRGD) was prepared for efficiently targeted delivery of antitumor drug, as shown in Figure 1. Stereocomplex interaction is one of the most effective approaches for improving the stability of PLA 5

ACCEPTED MANUSCRIPT micelles.[31,32] cRGD has a high affinity for the αvβ3 integrin receptor overexpressed on the surface of tumor vasculature and the membrane of some tumor cells, such as

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colorectal cancer cells, breast cancer cells, and prostate cancer cells, which render cRGD a promising molecule ligand for targeted chemotherapies of different

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cancers.[33] Herein, doxorubicin (DOX) was used as a model drug. As expected, both the DOX-loaded non-targeting and targeting PLA micelles, marked as SCM/DOX and

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cRGD-SCM/DOX, respectively, exhibited regular and stable spherical structure owing to the stereocomplex interaction between dextrorotatory and levorotatory PLA. Moreover, with the introduction of cRGD, the targeting micelle greatly improved the

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antitumor efficacy of DOX through the selectively recognizing tumor cells. The

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targeting multicomponent micelle with easily replaceable ligand might be a promising

Methods

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platform for selective delivery of antitumor drug into lesion site.

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Proton nuclear magnetic resonance (1H NMR) spectra were detected on a Bruker AV 600 NMR spectrometer (Billerica, MA, USA) in deuterochloroform (CDCl 3). The morphologies of non-targeting and targeting stereocomplex micelles (SCMs) were revealed by a transmission electron microscopy (TEM; JEM-1011, JEOL, Tokyo, Japan). The hydrodynamic diameters (Dhs) were detected by a dynamic laser scattering (DLS) measurements (WyattQELS instrument, DAWN EOS, Wyatt Technology Corporation, Santa Barbara, CA, USA) with a scattering angle at 90°. All other experimental protocols are described in detail in Supporting information. Results 6

ACCEPTED MANUSCRIPT Preparations and characterizations of drug-loaded micelles In this work, DOX was employed as a broad spectrum of anthracycline antitumor

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drug to verify the targeted sustained drug delivery capability of cRGD-SCM (Figure 1). In this nanoplatform, 4-arm poly(ethylene glycol)-block-poly(D-lactide) (4-arm

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PEG-b-PDLA), methoxy poly(ethylene glycol)-block-poly(L-lactide) (mPEG-b-PLLA), and c(RGDfC)-poly(ethylene glycol)-block-poly(L-lactide) (cRGD-PEG-b-PLLA) were

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used as polymer matrices. 4-arm PEG-b-PDLA was applied as a provider of dextrorotatory PLA moiety and also served as a cross-linker of SCM through stereocomplex interaction. cRGD-PEG-b-PLLA was used as a donor of targeting

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agent and levorotatory PLA segment, and mPEG-b-PDLA was employed for adjusting

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the density of targeting ligand. As depicted in Supplementary Figure S4, 4-arm PEG-b-PDLA was synthesized through the ring-opening polymerization (ROP) of

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D-LA with 4-arm PEG as a macroinitiator and stannous(II) 2-ethylhexanoate

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(Sn(Oct)2) as a catalyst. mPEG-b-PLLA and aPEG-b-PLLA with terminal modifiable allyloxy group were synthesized through the similar route as 4-arm PEG-b-PDLA with L-LA as an alternative monomer (Supplementary Figures S4 and S5). As shown in Supplementary Figure S5, c(RGDfC) was conjugated to the terminal of aPEG-b-PLLA through thiol-ene click reaction to obtain cRGD-PEG-b-PLLA. The chemical structures were confirmed by the thoroughly annotated proton resonance signals in 1

H NMR spectra (Supplementary Figures S4 and S5). The degrees of polymerization

(DPs) of DLA and LLA were both calculated to be 16. The non-targeting SCM was composed of 4-arm PEG-b-PDLA and mPEG-b-PLLA. DOX was loaded into the 7

ACCEPTED MANUSCRIPT non-targeting and targeting micelles through nanoprecipitation to prepare SCM/DOX and cRGD-SCM/DOX, respectively. The drug loading contents (DLCs) and drug

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loading efficiencies (DLEs) were calculated to be 8.9 ± 0.37 and 8.4 ± 0.29 wt.%, and 48.8 ± 2.64 and 45.9 ± 1.35 wt.%, respectively, through standard curve method.

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The properties were systemically revealed. Firstly, the morphologies and apparent sizes were demonstrated by TEM. As shown in Figure 2A, both SCM/DOX and

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cRGD-SCM/DOX exhibited a uniform spherical structure with the diameters of about 85 and 90 nm, respectively. As depicted in Figure 2B, the Dhs of SCM/DOX and cRGD-SCM/DOX were determined to be 91.6 ± 4.9 and 102.9 ± 5.6 nm by DLS,

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respectively. The smaller sizes observed from TEM compared to those determined by

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DLS may be due to the dehydration of micelles in the preparation process of TEM specimen.[34] The size of nanoparticle plays an important role in controlled drug

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delivery. When the size of nanoparticle is less than 50 nm, the nanoparticle widely

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distributes in the normal tissues and organs, resulting in high systemic toxicity and low tumor accumulation,[35] and on the other hand, it will be eliminated from plasma rapidly. Furthermore, when the size of nanoparticle is greater than 200 nm, it will be captured by reticuloendothelial system (RES).[36,37] It is well-known that the proper size of nanocarrier might improve the passive targeting accumulation in the tumor through the enhanced permeability and retention (EPR) effect.[38,39] The results indicated that the sizes of the SCMs were appropriate for drug delivery in vivo, in that the befitting size might prolong the circulation time and increase the selective tumor accumulation.[40,41] 8

ACCEPTED MANUSCRIPT The in vitro DOX release kinetics of SCM/DOX and cRGD-SCM/DOX were detected in phosphate-buffered saline (PBS) of pH 7.4, 37 °C. As shown in Figure 2C,

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a similar rapid DOX release of about 30% from SCM/DOX and cRGD-SCM/DOX was observed within 8 h. Subsequently, DOX was released in a steadier pattern, and

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approximately 50% of DOX was released at 72 h. The initial fast release might be attributed to the dissociation of surface-absorbed drug presented in the hydrophilic

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shell of micelle, while the sustained release was likely assigned to the slow release of DOX entrapped in the hydrophobic core of micelle.[34] It should be noted that about half of the encapsulated drug has not been released in the test duration, which may

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be associated with the hydrophobic core of micelle tightly, as observed previously.[42]

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The result indicated that the encapsulation of DOX in SCMs possessed the characteristic of continuous and prolonged release.

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Cell uptake and proliferation inhibition

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The murine C26 colon cancer cells were cultured for investigating the cell internalization of DOX-loaded SCMs and intracellular release of DOX. After treated with free DOX, SCM/DOX, and cRGD-SCM/DOX for 2 h, C26 cells were observed by confocal laser scanning microscopy (CLSM). Since DOX is autofluorescent, it was used directly to measure cell uptake without additional marker, and fluorescence intensity should be directly proportional to the amount of internalized DOX. [22] As shown in Figure 3A, the fluorescence of DOX could be observed in the nuclei of cells. The free DOX group exhibited the lowest DOX fluorescence intensity among all DOX formulation groups. The fluorescence intensity of DOX in the SCM/DOX group was 9

ACCEPTED MANUSCRIPT weaker

than

that

of

the

cRGD-SCM/DOX

group,

while

the

signal

of

cRGD+cRGD-SCM/DOX group was similarly as that of the SCM/DOX group. It might

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indicate that the cell uptake of cRGD-SCM/DOX was more efficient than that of SCM/DOX, owing to the quicker ligand-receptor mediated endocytosis of Moreover,

the

pretreatment

with

20.0

mM

free

cRGD

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cRGD-SCM/DOX.

downregulated the cell internalization of cRGD-SCM/DOX, and the finding confirmed

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its cRGD-enhanced endocytosis. As depicted in Figure 3B, fluorescence-activated flow cytometry (FCM) was used to further quantitatively assess the cell uptake of free DOX, SCM/DOX, and cRGD-SCM/DOX toward C26 cells without or with

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pretreatment of free cRGD. The results demonstrated that there was more cell

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internalization of cRGD-SCM/DOX compared with those of free DOX and SCM/DOX after treated for 2 h. As detected in CLSM assay, the pretreatment with free cRGD

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inhibited the ligand-receptor mediated endocytosis of cRGD-SCM/DOX. The

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interaction between cRGD ligand and αvβ3 integrin might be responsible for the difference in cell internalization of SCM/DOX and cRGD-SCM/DOX.[43] The expression of αvβ3 integrin was up-regulated on the surface of some tumor cells, but rarely or not in normal tissue. cRGD can bind to the over-expressed αvβ3 integrin on the membrane of C26 cells specifically, which may cause more cRGD-SCM/DOX to be attached to the tumor cells. Thus, the cell internalization of cRGD-SCM/DOX was enhanced compared with free DOX and SCM/DOX, which was considered as active targeting effect.[15] The in vitro cell proliferation inhibition efficacies of free DOX, SCM/DOX, and 10

ACCEPTED MANUSCRIPT cRGD-SCM/DOX against C26 cells were determined by a methyl thiazolyl tetrazolium (MTT) assay. The results showed that cRGD-SCM/DOX exhibited a better

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proliferation inhibition effect as compared to those of free DOX and SCM/DOX at both 24 and 48 h (Figure 3C). In addition, the difference among cRGD-SCM/DOX,

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SCM/DOX, and free DOX became more significant at lower equivalent concentration of DOX. In addition, the half-maximal inhibitory concentrations (IC50s) of free DOX,

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SCM/DOX, and cRGD-SCM/DOX were calculated and listed in Table S1. The differences between any two groups of IC50 were statistically significant (P < 0.05). All the findings might be attributed to the over-expressed αvβ3 integrin on the membrane

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of C26 cells, which led to the enhanced internalization of cRGD-SCM/DOX. The

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results were consistent with the efficiencies of cell uptake, which were determined from CLSM and FCM assays.

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Pharmacokinetics and tissue distribution

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In the present study, the blood clearance of various DOX formulations was evaluated by high performance liquid chromatography (HPLC, Waters Co., Milford, MA, USA) to detect the different circulation durations of free DOX, SCM/DOX, and cRGD-SCM/DOX. As shown in Figure 4A, the free DOX in the plasma was rapidly cleared from blood circulation during a very short period, and DOX could hardly be detected in the plasma at 1 h after injection. In contrast, the DOX concentrations of SCM/DOX and cRGD-SCM/DOX decreased slowly, which indicated an obvious retardation in clearance from blood. In detail, the elimination half-lives (t1/2s) of free DOX, SCM/DOX, and cRGD-SCM/DOX were calculated to be 0.25, 2.67, and 1.73 h, 11

ACCEPTED MANUSCRIPT respectively, using PKSolver software (version 2.0; China Pharmaceutical University, Nanjing, P. R. China), as listed in Supplementary Table S2. The results confirmed the

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extended circulation times of DOX-loaded SCMs, especially SCM/DOX. The extended circulation times of DOX-loaded SCMs should be assigned to their

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properties of anti-protein adsorption and clearance evasion of the reticuloendothelial system (RES) as a benefit of the outer PEG shell. Moreover, both SCMs exhibited

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significantly increased area under the curve (AUC0-t) in blood, that is, 1.60 and 1.58 times higher than free DOX, owing to the "stealth" effect induced by the PEGylation. To evaluate the targeting efficiency of cRGD-modified SCM in vivo, the ex vivo

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fluorescence imaging of isolated visceral organs (i.e., the heart, liver, spleen, lung,

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and kidney) and tumors at 2 and 12 h post-injection were carried out in the C26 tumor-grafted mice. As shown in Figure 4B, the liver and kidney showed the strongest

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DOX fluorescence in all the test groups and time durations, indicating that the

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majority of each DOX formulation was captured and metabolized by the liver and kidney.[22] Compared with that of the free DOX group, a fairly weaker fluorescence in the liver and kidney were observed in the groups of DOX-loaded SCMs, due to the rapid in vivo metabolism of free DOX. The decreased accumulation of DOX-loaded SCMs might down-regulate the toxicity to both the liver and kidney. In addition, at 12 h post-injection, the fluorescence intensities of various organs were decreased over time in all the three groups. More interestingly, the tumor of cRGD-SCM/DOX group showed the significantly strongest fluorescence intensity than the tumors of free DOX and SCM/DOX groups, owing to the enhanced accumulation of cRGD-SCM/DOX 12

ACCEPTED MANUSCRIPT through the synergistic targeting of the EPR effect and cRGD−αvβ3 integrin recognition. The tumor in the free DOX group showed the weakest fluorescence

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intensity at 12 h post-injection because of the poor accumulation of free DOX in tumor tissue. And then, the photon numbers per unit area (i.e., average signals) of the

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groups of various formulations were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the results were shown in Figure 4C. The

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average signal of the tumor in the cRGD-SCM/DOX group at 12 h post-injection was 1.37 and 1.31 times higher than those in the free DOX and SCM/DOX groups, respectively. The results semi-quantitatively indicated that cRGD-SCM/DOX

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significantly improved the selective accumulation of DOX in tumor. This could be

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explained from two aspects: on the one hand, the appropriate size of cRGD-SCM/DOX might contribute to the enhanced accumulation in the tumor tissue

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by the EPR effect;[35] on the other hand, the interaction between RGD ligand and αvβ3

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integrin expressed on the membrane of endothelial cells in the vascular of tumor tissue or on the surface of tumor cells might also lead to the enhanced accumulation in tumor tissue.[43]

Antitumor efficacy and security in vivo In vivo antitumor efficacy is one of the most important indicators for evaluating the advanced antineoplastic agents. C26 colon cancer-grafted BALB/c mouse model was constructed to evaluate the antitumor efficacies of various DOX formulations. Typically, normal saline (NS) as a control, and free DOX, SCM/DOX, and cRGD-SCM/DOX at a DOX dose of 5.0 mg per kg body weight (mg (kg BW)−1) have 13

ACCEPTED MANUSCRIPT been administrated via intravenous injection every four days for a total of five injections, since the tumor volumes reached to approximately 95 mm3. As depicted in

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Figure 4D, the tumor volumes of various groups at each point were measured every two days. The results demonstrated that the treatments with free DOX, SCM/DOX,

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and cRGD-SCM/DOX more significantly suppressed the growth of tumors compared with NS as a control. Especially, the mean tumor volume of cRGD-SCM/DOX was

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lower than those of free DOX and SCM/DOX after four times of treatments, i.e., from day 13, indicating the excellent antitumor efficacy of cRGD-SCM/DOX. The most efficient antitumor efficacy of cRGD-SCM/DOX might be attributed to the highly

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selective accumulation of targeting DOX-loaded SCM in tumor tissue through the

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combination of the EPR effect and the targeted property of cRGD ligand. In addition, somewhat less antitumor capability of free DOX should be assigned to its rapid

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clearance from circulation.

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In order to further evaluate the antitumor efficacies of different DOX formulations, the tumors were isolated and collected after the mice were sacrificed on day 19, and sectioned for histopathology analyses. As shown in Figure 5A, tumor cells with large hyperchromatic pleomorphic nuclei were observed in each tumor tissue sections of different groups after hematoxylin and eosin (H&E) staining. In the free DOX, SCM/DOX, and cRGD-SCM/DOX groups, different levels of tumor necrosis were observed, such as nuclear shrinkage, fragmentation, or absence. In addition, as depicted in Figure 5B, the necrosis area in the cRGD-SCM/DOX group was 1.99 and 1.4 times higher than those of free DOX and SCM/DOX groups as calculated using 14

ACCEPTED MANUSCRIPT NIS Elements BR Imaging software (Nikon Instruments SpA, Florence, Italy), indicating the greatest antitumor potential of cRGD-SCM/DOX. The differences of

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therapeutic effects after injection of various DOX formulations were further confirmed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate

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nickend labeling (TUNEL) assay. Some types of chemotherapy drugs like DOX would cause the fracture of DOX in the tumor cells, and then the DNA fragmentation can be

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dyed with green fluorescence by a fluorescein isothiocyanate (FITC)-marked TUNEL kit. As shown in Figure 5A, different levels of punctiform and flake green fluorescent signals were observed in all treated tumor sections with various DOX formulations.

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Wider and stronger fluorescence signal indicates more cell apoptosis in tumor tissue.

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The highest level of cell apoptosis was observed in the cRGD-SCM/DOX group. However, the apoptotic cells could hardly be observed in the tumor treated by NS as a

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control. The semi-quantitative analyses demonstrated that cRGD-SCM/DOX induced

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1.88 and 1.66 times cell apoptosis than free DOX and SCM/DOX, respectively, as analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The results confirmed the advantage of cRGD-SCM/DOX in the chemotherapy of colon cancer. In addition, the immunofluorescence analyses of Ki-67 and cleaved poly (ADP-ribose) polymerase (cPARP) were processed to reveal various degrees of cell apoptosis in the tumor sections treated with NS as a control and different DOX formulations. Ki-67 and cPARP are a pair of opposite markers, which indicates cell proliferation and apoptosis, respectively. As depicted in Figure 5A, in contrast with free DOX and SCM/DOX, the tumor treated with cRGD-SCM/DOX showed the lowest 15

ACCEPTED MANUSCRIPT fluorescent signal of Ki-67, which reflected the most active cell proliferation, and the highest fluorescent signal of cPARP, which demonstrated the most severe cell

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apoptosis. Simi-quantitatively analyzed by ImageJ software, the cRGD-SCM/DOX group exhibited 0.33- and 0.37-fold fluorescent signal of Ki-67, and 1.83 and 1.54

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times higher fluorescent signal of cPARP than free DOX and SCM/DOX. The results demonstrated that there were the most tumor cells underwent apoptosis in the

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cRGD-SCM/DOX group compared with those of the free DOX and SCM/DOX groups. In summary, all the findings of H&E, TUNEL, Ki-67, and cPARP were consistent with the inhibition curve of tumor growth, as depicted in Figure 4D, which all revealed the

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greatest antitumor efficacy of cRGD-SCM/DOX.

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Because the tumor suppression of antitumor drug is always accompanied by a variety of side effects, the security assessments play an important role in clinical

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application.[14,18,27] In order to evaluate the safety of various DOX formulations, the

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major organs (i.e., heart, liver, spleen, lung, and kidney) were harvested from mice on day 2 after the last injections, and then sliced and stained by H&E for histopathology analyses. The organs of control group showed relatively normal histological structure (Figure 6). However, the heart sections of all groups treated with various DOX formulations showed different degrees of myocardial injury, such as edema of myocardial cells, irregular arrangement of myocardial cells, and infiltration of inflammatory cells. In addition, pathological changes of the lungs could also be observed in the groups of free DOX and DOX-loaded SCMs, for instance, thickening or blocking of alveolar walls. In short, the damage levels of the organs in the free 16

ACCEPTED MANUSCRIPT DOX group were higher than those of the SCM/DOX and cRGD-SCM/DOX groups. Nevertheless, no significant morphological changes could be detected for all other

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organs in the groups of both DOX-loaded SCMs. The results suggested a high biosecurity of DOX-loaded SCMs.

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Discussion

The targeting micelles have been extensively studied for the selective delivery of

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antitumor drugs to significantly upregulate the efficacy and decrease the side effects preclinically and even clinically.[24] However, the difficulty in targeting ligand replacement and insufficient stability limits the convenient applications of micelles in

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directional drug delivery toward different types of cancers.[32,44]

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In order to solve the above problem, a multicomponent PLA SCM composed of 4-arm PEG-b-PDLA, mPEG-b-PLLA, and cRGD-PEG-b-PLLA was designed and

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prepared in this study for targeted delivery of DOX, which was marked as

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cRGD-SCM/DOX (Figure 1). In this platform, the targeting component of cRGD-PEG-b-PLLA can be facilely replaced by other targeting ligands-modified PEG-b-PLLA according to the different types of receptors on the membranes of tumor cells. Moreover, the stereocomplex interaction between PLLA and PDLA segments in the core can effectively improve the stability of micelle.[45] cRGD-SCM/DOX showed a spherical morphology with a diameter of around 100 nm (Figure 2). The moderate size facilitates the improved accumulation of micelle through the EPR effect.[38,39] The targeted drug-loaded micelle could be uptaken by C26 cells and inhibited cell proliferation more efficiently compared with free DOX and non-targeting system 17

ACCEPTED MANUSCRIPT (Figure 3). The results should be attributed to the specific recognition of cRGD in cRGD-SCM/DOX and αvβ3 integrin over-expressed on the membrane of C26 cells.

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More fascinatingly, cRGD-SCM/DOX exhibited extended circulation time, selective accumulation in tumor tissue, and enhanced antitumor efficacy toward αvβ3

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integrin-positive C26 colon cancer in comparison with free DOX and even non-targeting SCM/DOX in vivo (Figure 4). The histopathological (H&E), in situ cell

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apoptosis (TUNEL), and immunofluorescence (Ki-67 and cPARP) analyses were further used to qualitatively and semi-quantitatively demonstrate the superiority of targeted DOX-loaded SCM in tumor suppression (Figure 5). Furthermore, the safety

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of cRGD-SCM/DOX for in vivo application was confirmed by the histopathological

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analysis of main organs (i.e., the heart, liver, spleen, lung, and kidney) from C26-grafted mice after treatments with various DOX formulations (Figure 6). The

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findings reveal that cRGD-SCM exhibits great potential in upregulating efficacy and

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decreasing toxicity of free drugs in vivo. Of course, the targeting SCM could be employed to selectively deliver other chemotherapeutic agents. In view of the above excellent properties, the targeting SCM might have great potential in personalized chemotherapy of different malignancies in clinic.

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Figure legends

Figure 1. Schematic illustration of multicomponent PLA SCM for selectively

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intracellular sustained drug release.

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Figure 2. Properties of SCM/DOX and cRGD-SCM/DOX. (A) TEM micrographs, (B) Dhs, and (C) release profiles of SCM/DOX and cRGD-SCM/DOX in PBS at pH 7.4.

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Figure 3. Cell uptakes and proliferation inhibition of various DOX formulations. (A) CLSM microimages and (B) FCM analyses for cell internalization of free DOX,

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SCM/DOX, and cRGD-SCM/DOX after incubation with C26 cells for 2 h. (C) In vitro

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cytotoxicity of free DOX, SCM/DOX, and cRGD-SCM/DOX after incubation with C26 cells for 24 and 48 h. Scale bars: 20.0 μm. Statistical data are presented as a mean ± standard deviation (SD; n = 6; #P < 0.01, &P < 0.001).

Figure 4. In vivo pharmacokinetics, tissue distribution, and C26 colon cancer inhibition. (A) Concentration-time profiles of DOX in plasma of male Sprague-Dawley rats after a single intravenous administration of free DOX, SCM/DOX, or cRGD-SCM/DOX. (B) Ex vivo DOX fluorescence images indicating tissue distribution of DOX, and (C) average signals collected from the major organs (i.e., the heart, liver 24

ACCEPTED MANUSCRIPT spleen, lung, and kidney) and tumor after caudal vein injection of free DOX, SCM/DOX, or cRGD-SCM/DOX to C26 subcutaneous grafted mice for 2 and 12 h. (D)

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Tumor volume of C26-grafted mice after treatment with NS as a control, free DOX, SCM/DOX, or cRGD-SCM/DOX. Statistical data are presented as mean ± SD (n = 3

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cRGD-SCM/DOX. Scale bars: 75.0 μm. (B) Necrotic area of tumor sections from H&E, (C) apoptosis area of tumor sections from TUNEL, (D) proliferation area of tumor

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sections from Ki-67, and (E) apoptosis area of tumor sections from PARP. Statistical

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data are presented as a mean ± SD (n = 3; *P < 0.05).

Figure 6. Security in vivo. Ex vivo histopathological (H&E) analyses of main organ (i.e., the heart, liver, spleen, lung, and kidney) sections from C26-grafted mice after treatment with NS as a control, free DOX, SCM/DOX, or cRGD-SCM/DOX. Scale bars: 75.0 μm.

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Figure 2. Properties of SCM/DOX and cRGD-SCM/DOX. (A) TEM micrographs, (B)

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Dhs, and (C) release profiles of SCM/DOX and cRGD-SCM/DOX in PBS at pH 7.4.

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Figure 3. Cell uptakes and proliferation inhibition of various DOX formulations. (A) CLSM microimages and (B) FCM analyses for cell internalization of free DOX, SCM/DOX, and cRGD-SCM/DOX after incubation with C26 cells for 2 h. (C) In vitro cytotoxicity of free DOX, SCM/DOX, and cRGD-SCM/DOX after incubation with C26 cells for 24 and 48 h. Scale bars: 20.0 μm. Statistical data are presented as a mean ± standard deviation (SD; n = 6; #P < 0.01, &P < 0.001).

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Figure 4. In vivo pharmacokinetics, tissue distribution, and C26 colon cancer

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inhibition. (A) Concentration-time profiles of DOX in plasma of male Sprague-Dawley rats after a single intravenous administration of free DOX, SCM/DOX, or

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cRGD-SCM/DOX. (B) Ex vivo DOX fluorescence images indicating tissue distribution of DOX, and (C) average signals collected from the major organs (i.e., the heart, liver spleen, lung, and kidney) and tumor after caudal vein injection of free DOX, SCM/DOX, or cRGD-SCM/DOX to C26 subcutaneous grafted mice for 2 and 12 h. (D) Tumor volume of C26-grafted mice after treatment with NS as a control, free DOX, SCM/DOX, or cRGD-SCM/DOX. Statistical data are presented as mean ± SD (n = 3 for A and C, n = 6 for D; *P < 0.05, #P < 0.01, &P < 0.001).

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immunofluorescence analyses. (A) Histopathological (H&E), in situ cell apoptosis (TUNEL), and immunofluorescence (Ki-67 and cPARP) analyses of tumor tissue sections after treatment with NS as a control, free DOX, SCM/DOX, or cRGD-SCM/DOX. Scale bars: 75.0 μm. (B) Necrotic area of tumor sections from H&E, (C) apoptosis area of tumor sections from TUNEL, (D) proliferation area of tumor sections from Ki-67, and (E) apoptosis area of tumor sections from PARP. Statistical data are presented as a mean ± SD (n = 3; *P < 0.05).

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Figure 6. Security in vivo. Ex vivo histopathological (H&E) analyses of main organ (i.e., the heart, liver, spleen, lung, and kidney) sections from C26-grafted mice after treatment with NS as a control, free DOX, SCM/DOX, or cRGD-SCM/DOX. Scale bars: 75.0 μm.

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Polylactide stereocomplex micelles (PLA SCMs) exhibit great potential for sustained drug delivery as a benefit of their improved stability. In this study, a cRGD-decorated PLA SCM (cRGD-SCM) was designed and prepared for targeted treatment of αvβ3 integrin-positive C26 colon cancer. The doxorubicin-loaded cRGD-SCM exhibited extended circulation time, selective accumulation in tumor tissue, and enhanced antitumor efficacy. In addition, the targeting component can be facilely replaced by other targeting ligands according to the different types of receptors on the membrane of tumor cells. All in all, the targeted SCM might have great potential in personalized chemotherapy of different malignancies clinically.

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