Tumor-specific penetrating peptides-functionalized hyaluronic acid-d -α-tocopheryl succinate based nanoparticles for multi-task delivery to invasive cancers

Accepted Manuscript Tumor-specific penetrating peptides-functionalized hyaluronic acid-D-α-tocopheryl succinate based nanoparticles for multi-task delivery to invasive cancers De-Sheng Liang, Hai-Tao Su, Yu-Jie Liu, Ai-Ting Wang, Xian-Rong Qi PII:

S0142-9612(15)00701-2

DOI:

10.1016/j.biomaterials.2015.08.035

Reference:

JBMT 17030

To appear in:

Biomaterials

Received Date: 24 February 2015 Revised Date:

15 August 2015

Accepted Date: 18 August 2015

Please cite this article as: Liang D-S, Su H-T, Liu Y-J, Wang A-T, Qi X-R, Tumor-specific penetrating peptides-functionalized hyaluronic acid-D-α-tocopheryl succinate based nanoparticles for multi-task delivery to invasive cancers, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.08.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Tumor-specific penetrating peptides-functionalized hyaluronic acid-D-α-tocopheryl succinate

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based nanoparticles for multi-task delivery to invasive cancers

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De-Sheng Liang1,2, Hai-Tao Su1, 2, Yu-Jie Liu1,2, Ai-Ting Wang1,2, Xian-Rong Qi1,2,3,*

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Molecular Pharmaceutics and New Drug Delivery System, 3State Key Laboratory of Natural

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and Biomimetic Drugs, 38 Xueyuan Road, Haidian District, Beijing 100191, PR China

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*Corresponding author: Xian-Rong Qi, School of Pharmaceutical Sciences, Peking

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University, 38 Xueyuan Road, Beijing, 100191, China Tel. & Fax: (+) 86 10 82801584

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E-mail: [email protected], [email protected]

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Beijing Key Laboratory of

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School of Pharmaceutical Sciences, Peking University,

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Abstract Poor site-specific delivery and incapable deep-penetration into tumor are the intrinsic

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limitations to successful chemotherapy. Here, the tumor-homing penetrating peptide

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tLyP-1-functionalized nanoparticles (tLPTS/HATS NPs), composed of two modularized

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amphiphilic conjugates of tLyP-1-PEG-TOS (tLPTS) and TOS-grafted hyaluronic acid

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(HATS), had been fabricated for tumor-targeted delivery of docetaxel (DTX). The prepared

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tLPTS/HATS NPs had about 110 nm in mean diameter, high drug encapsulation efficiency

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(93%), and sustained drug release behavior. In vitro studies demonstrated that the

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tLPTS/HATS NPs exhibited enhanced intracellular delivery and much better anti-invasion

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ability, cytotoxicity, and apoptosis against both invasive PC-3 and MDA-MB-231 cells as

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compared to the non-tLyP-1-functionalized HATS NPs. The remarkable penetrability and

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inhibitory effect on both PC-3 and MDA-MB-231 multicellular tumor spheroids were also

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identified for the tLPTS/HATS NPs. In vivo biodistribution imaging demonstrated the

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tLPTS/HATS NPs possessed much more lasting accumulation and extensive distribution

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throughout tumor regions than the HATS NPs. The higher in vivo therapeutic efficacy with

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lower systemic toxicity of the tLPTS/HATS NPs was also verified by the PC-3 xenograft

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model in athymic nude mice. These results suggested that the designed novel tLPTS/HATS

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NPs were endowed with tumor recognition, internalization, penetration, and anti-invasion,

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and thus might be a promising anticancer drug delivery vehicle for targeted cancer therapy.

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Key words: tumor-homing penetrating peptide tLyP-1; site-specific delivery; NRP-1

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receptor; CD44 receptor; D-α-tocopheryl succinate (TOS)

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1. Introduction Efficient and site-specific delivery of anticancer drugs into tumors presents a critical

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challenge for the success of cancer chemotherapy [1]. Currently, several of conventional

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nanocarriers (e.g., liposomes, micelles, and polymeric nanoparticles, etc.) have progressed

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greatly and offered an available cancer-targeted treatment via the leaky tumor vasculature,

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termed enhanced permeability and retention (EPR) effect [2, 3]. And some of them have

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been applied in preclinical or in clinical trials. However, an adequate treatment remains to

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be elusive due to their poor tumor site-specific delivery and tumor tissue penetration. The

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development of a multi-task delivery platform for more effectively targeted treatment is

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therefore still urgently required [4]. Over the past decades, a variety of different strategies

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have been proposed to improve the targeted delivery and intracellular internalization of

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nanocarriers by conjugation of ligands or antibodies. The ligand-conjugated nanocarriers

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directly interact with complementary receptors present on the surface of target cells [5-8].

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The major benefit of the actively targeted nanocarriers over those passively targeted ones is

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that they can be enriched within tumors for a longer period of time because of their binding

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to or their uptake by cancer cells, preventing them from rapidly re-entering systemic

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circulation [9].

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The penetration into the inner tumor tissue to deliver sufficient anticancer drugs is

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important to the efficacy of the actively targeted nanocarriers. However, the disordered

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tumor vascular system, deregulated extracellular matrix and high interstitial pressure in the

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abnormal tumor microenvironment cooperatively hinder the drug distribution into tumor

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region. This is a formidable barrier to cancer chemotherapy and a driving force leading to

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tumor recurrence, progression and mutidrug resistance [10, 11]. In light of this difficulty,

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the cell penetrating peptides (CPPs)-modified nanocarriers offers an alternative strategy to

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address the incapable tumor tissue penetration. Unfortunately, the majority of the known

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CPPs were devoid of tissue-selectivity, readily to be contacted and internalized by nearly

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all cell types, which in turn accelerated in vivo plasma clearance, and thereby lessening the

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therapeutic response [12, 13]. The Cend Rule (CendR, (R/K)XX(R/K)) motif peptides were reported to bear

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tumor-specific penetration via a mechanism of ligand-receptor recognition [14, 15]. The

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receptor for the CendR motif is neuropilin (NRP-1), which is a modular transmembrane

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protein overexpressed on the surface of a wide range of cancers, while a relatively low

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expression in normal tissues [16-18]. The tLyp-1 peptide (CGNKRTR), identified as a

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substrate for NRP-1 receptor with high affinity and specificity, contains both a

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tumor-homing motif and a cryptic CendR, which can induce tumor cell recognition and

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tissue penetration through NRP-dependent internalization pathway [19, 20]. This

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promising feature makes the tLyp-1 peptide as an effective targeting moiety to mediate

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tumor site-specific delivery and penetration of nanocarriers into solid tumor.

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Nanocarriers functionalized with biological molecules may trigger additive therapeutic

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effect with the delivered drugs after intracellular uptake. One typical example is

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D-α-tocopheryl derivatives-based nanocarriers for anticancer drug delivery [21, 22]. The two

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D-α-tocopheryl

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polyethylene glycol-succinate (TPGS), exert highly selective antitumor effect by specifically

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destabilizing cancer cell mitochondria but nontoxic toward normal cells [23-26]. TOS has

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been grafted on a variety of polymers to allow for better encapsulation and delivery of poorly

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water soluble drugs. Both TOS-grafted chitosan copolymer and TOS-modified pluronic P123

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copolymer demonstrated high drug loading capacity for paclitaxel, and excellent in vivo

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circulation property [27, 28]. The synergic capabilities of TOS to act with the delivered

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chemotherapeutics have also been verified in vitro and in vivo for the TOS-based

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nanocarriers [26, 29].

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D-α-tocopheryl

succinate

(TOS)

and

D-α-tocopheryl

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ACCEPTED MANUSCRIPT Here, an active tumor-targeted hyaluronic acid (HA) and TOS-based nanoparticle delivery

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system was rationally designed using tLyp-1 peptide as targeting moiety. HA bears specific

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binding property to cancer cells overexpressing CD44 receptor and its derivatives have been

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extensively utilized as nanocarriers for tumor-targeted drug delivery [30, 31]. We first

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attempted to build the amphiphilic targeting molecule, tLyP-1-PEG-TOS (tLPTS), by

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chemical linking the TOS to tLyp-1 peptide via the hydrophilic polyethylene glycol (PEG)

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chain. This ligand-PEG-lipid conjugate was widely utilized as active-targeting components in

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the targeted nanocarriers design [32-35]. Besides, the amphiphilic TOS-grafted HA

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copolymer (HATS) was constructed by the hydrophobic TOS chemically conjugated to the

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backbone of HA. Thus, a multifunctional nanoparticle delivery platform (tLPTS/HATS NPs)

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was fabricated by simply mixing two conjugates of tLPTS and HATS (Scheme 1). The PEG

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and tLyP-1, located on the outer layer, was for extended blood circulation and active

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tumor-targeting, respectively, while the TOS was for synergistic effect with the delivered

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therapeutics of docetaxel (DTX). Nanocarriers functionalized with elements related to tumor

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cell-surface molecules are susceptible to tumor cell recognition and the ensuing transcytosis,

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which has been demonstrated to be an attractive choice to overcome the sequential delivery

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barriers [36, 37]. But no research that we know has reported using biologically functional HA

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and CendR motif peptides tLyP-1 to develop a nanoparticle delivery system for more

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effectively targeted treatment of solid tumor. It was assumed that a combination of CD44

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targeting and tumor lineage-homing penetrating peptides of tLyP-1 may contribute to tumor

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site-specific accumulation and penetration into tumor. The objectives of this study were to

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evaluate the potential of the developed tLPTS/HATS NPs to improve the anticancer drug

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delivery and therapeutic efficacy and to explore the underlying mechanisms.

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Scheme 1. (A) Schematic representation of DTX-loaded tLPTS/HATS NPs. (B) Schematic

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illustration of tumor-targeted delivery strategy for the tLPTS/HATS NPs. After prolonged

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blood circulation by composite hydrophilic shell of HA and PEG, the tLPTS/HATS NPs

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passively accumulated at tumor site via EPR effect, followed by promoted intracellular

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delivery and deep penetration into tumor tissues through CD44 and NRP-1 dual

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receptor-mediated transcytosis.

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2. Materials and methods 2.1. Materials Hyaluronic acid (HA, 7.8 kDa) was obtained from Shandong Freda Biochem Co., Ltd. 6

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(Shandong, China). tLyP-1 peptide (CGNKRTR, MW 833.97) was synthesized by GL

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Biochem. (Shanghai, China). Maleimide-PEG2000-NH2 was purchased from JenKem

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Technology Co., Ltd. (Beijing, China). Docetaxel (DTX) was obtained from Norzer

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Pharmaceutical Co., Ltd. Taxotere® was commercially available from the local hospital of

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Beijing

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Sigma-Aldrich

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Co.

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N-Hydroxysuccinimide (NHS) were purchased from Sinopharm Group Co., Ltd. (Beijing,

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China) and Advanced Chem. Tech. Co., Ltd. (Shenzhen, China), respectively. Anhydrous

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formamide, anhydrous dimethylformamide (DMF) were obtained from Beijing Chemical

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Reagent Co., Ltd. (Beijing, China). Coumarin-6 (COU) and DiR fluorescent probes were

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purchased from Life Technologies (Eugene, OR, USA). Lyso-Tracker Red was obtained from

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Invitrogen (Carlsbad, CA, USA). Matrigel was purchased from BD Biocoat (Franklin, NJ,

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USA). Crystal violet was purchased from Amresco (Solon, OH, USA). Ham's F12 medium,

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Leibovitz's L15 medium, penicillin-streptomycin, trypsin and Hoechst 33258 were provided

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by Macgene Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was obtained from

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Invitrogen/Gibco (Grand Island, NY, USA). Annexin V-FITC apoptosis detection kit was

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purchased from Nanjing KeyGen Biotech. Co., Ltd (Nanjing, China). All other reagents and

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chemicals were of analytical grade and obtained from commercial sources.

Louis,

MO,

S.A.,

France).

USA).

D-α-tocopheryl

succinate

(TOS),

(MTT),

and

bromide

N,N'-dicyclohexylcarbodiimide

(DCC)

and

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(Aventis

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The male BALB/c nude mice (16−18 g) were provided by the Vital Laboratory Animal

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Center (Beijing, China). All care of the animals were performed under specific pathogen free

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(SPF) conditions with free access to standard food and water, and all the animal experiments

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were conducted with the approval of the Ethics Committee of Peking University.

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2.2. Synthesis of tLPTS and HATS conjugate The amphiphilic targeting molecules of tLyP-1-PEG-TOS (tLPTS) were synthesized by

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two-step procedure as illustrated in Fig. 1A. First, the carboxyl group of TOS (0.025 mmol)

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was activated with EDC (0.05 mmol) and NHS (0.05 mmol) in 2 mL of DMF for 8 h,

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followed by the addition of Maleimide-PEG2000-NH2 (0.025 mmol). After stirring in dark for

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another 8 h, the reaction mixture was dialyzed against distilled water for 48 h (MWCO 7000

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Da).

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Maleimide-PEG-TOS. Then, the targeting moiety of tLyP-1 peptide was conjugated to

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Maleimide-PEG-TOS by sulfydryl-maleimide coupling reaction. Briefly, in the presence of

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triethylamine (TEA, 15 µL), the tLyP-1 peptides (0.01 mmol) were added to the

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Maleimide-PEG-TOS (0.01 mmol) in 2 mL of DMF. After stirring for 8 h under nitrogen gas,

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the reaction mixture was dialyzed against distilled water for 48 h (MWCO 7000 Da). The

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purified tLPTS conjugate was obtained after lyophilization and stored at -20 °C. The

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chemical structure of the intermediate Maleimide-PEG-TOS and the final tLPTS conjugate

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were determined by 1H NMR analysis (400 MHz) and MALDI-TOF MS analysis.

residue

was

filtrated

and

lyophilized

to

obtain

the

intermediate

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The amphiphilic TOS-grafted hyaluronic acid copolymer (HATS) was synthesized by a

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coupling reaction of HA and aminated α-TOS (Fig. 1B). In brief, the TOS (0.6 mmol) was

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firstly converted to aminated TOS (TOS-NH2) by the excess of ethylenediamine (EDA,

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6mmol). Then, the TOS-NH2 was chemically conjugated to the backbone of HA (0.3 mmol)

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in the presence of EDC (0.6 mmol) and NHS (0.6 mmol) dissolved in 10 mL of formamide.

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The reaction mixture was stirred at room temperature for 24 h under nitrogen gas. The crude

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product was precipitated by excess ice-cold acetone and purified with another two washes.

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The resulting product was then dialyzed against distilled water for 48 h using a dialysis

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membrane (MWCO 3500 Da). The residue was filtrated and lyophilized to obtain the HATS

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conjugate as white floccules. The chemical structure of HATS conjugate were determined by

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H NMR analysis dissolved in D2O (400 MHz).

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2.3. Preparation and characterization of DTX-loaded HATS NPs and tLPTS/HATS NPs The DTX-loaded HATS NPs and tLPTS/HATS NPs were prepared by a modified

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emulsification−solvent evaporation method [26, 38]. Briefly, 10 mg of HATS conjugate

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mixed with a certain amount of tLPTS (0%, 10%, and 25% w/w, respectively) was dispersed

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in 4 mL of distilled water. Then, 1 mg of DTX in 200 µL of chloroform was added and

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sonicated at 100 W for 5 min in an ice bath with a probe-type ultrasonicator (JY92-2D;

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Ningbo Scientz Biotech. Co., Ltd., Ningbo, China). The resulting emulsion was evaporated

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overnight to obtain the nanoparticle suspensions. To remove the unloaded DTX, the

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suspensions were filtered through 0.45 µm pore size membrane, and the filtrate stored at 4 °C

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for use. The dye-labeled NPs (COU- or DiR-loaded NPs) were prepared with the same

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procedure except that DTX was replaced by COU or DiR, respectively. The modification

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ratio of tLPTS was set to 25% for the prepared tLPTS/HATS NPs if no special instructions.

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The particle size, polydispersity index (PDI) and zeta potential of NPs were measured by

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dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Zetasizer 3000HS,

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Malvern, Worcestershire, UK) at 25 °C. The transmission electron microscope was also used

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to visualize the morphology of the DTX-loaded NPs (TEM, JEOL, JEM-200CX, Japan).

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After dilution with distilled water, droplets of the nanoparticle suspensions were placed on

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the surface of a Formvar-coated copper grid, followed by a negative staining method using

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uranyl acetate solution (1%, w/v), and air-dried overnight at room temperature. To measure

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the encapsulation efficiency (EE %) and drug loading (DL %), the NPs were disrupted with

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methanol and the drug content in the nanoparticles (Wdrug in NPs) was determined by the HPLC

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method [26].

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2.4. DTX release from NPs in vitro The in vitro release profile of DTX from nanoparticles was examined by a dialysis method.

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The phosphate-buffered saline (PBS, pH 7.4) at 37 °C was used as the release medium with

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0.5% (w/v) Tween 80 to fit the sink condition. Briefly, 0.5 mL of DTX-loaded HATS NPs

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and tLPTS/HATS NPs was added into a dialysis bag (MWCO 3500 Da) and immersed in 50

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mL of release medium under stirring at 100 rpm. At designated time point, 0.5 mL of sample

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was collected and replaced with an equal volume of fresh release medium. The released

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amount of DTX was determined by HPLC after filtration through 0.22 µm membrane filter.

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2.5. Cytotoxicity of DTX-loaded NPs

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The human prostate cancer cells (PC-3) and breast cancer cells (MDA-MB-231) were

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provided by the Institute of Basic Medical Sciences, Peking Union Medical College. The

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PC-3 cells were cultured in Ham's F12 medium and MDA-MB-231 cells in Leibovitz's L15

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medium. Both the cell culture media were supplemented with 10% (v/v) FBS, 1% (v/v)

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penicillin (0.1 mg/mL) and streptomycin (0.1 mg/mL) in a 5% CO2 atmosphere and 95%

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relative humidity at 37 °C.

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The cytotoxicity of various DTX-loaded NPs against both PC-3 and MDA-MB-231 cells

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were assessed by the MTT-based assay. Briefly, PC-3 cells or MDA-MB-231 cells were

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seeded in 96-well plates at a density of 1 × 104 cells/well. After incubation for 24 h, the cells

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were treated with free DTX, Taxotere®, DTX-loaded NPs (HATS NPs, 10%- and

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25%-tLPTS/HATS NPs) at a series of DTX concentrations (from 1 ng/mL to 5 µg/mL) for 48

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h at 37 °C. Then, 20 µL of MTT (5 mg/mL in PBS) was added to each well. After incubation

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for 4 h, the culture medium were discarded and added with 200 µL of DMSO to dissolve the

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formazan. The absorbance of each well was measured by an iMark microplate reader

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(Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 570 nm.

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2.6. Cellular uptake studies of dye-labeled NPs The cellular uptake characteristics of the developed nanoparticles were evaluated by flow

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cytometry. Briefly, the two types of cells (PC-3 or MDA-MB-231 cells) were seeded in

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6-well culture plates at 4 × 105 cells/well and allowed to attach overnight. Then, the cells

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were exposed to free COU, COU-loaded HATS and tLPTS/HATS NPs for 2 h under 5% CO2

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at 37 °C. The final COU concentration was 0.5 µg/mL. After incubation for 2 h, the cells

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were harvested, rinsed with cold PBS, resuspended in 500 µL of PBS. In the competitive

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receptor study, the cells was pre-incubated with excess free HA or tLyP-1 (1 mM) for 1 h

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before adding tLPTS/HATS NPs. The cells fluorescence was determined by FACScan flow

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cytometry (Becton Dickinson, San Jose, CA, USA)

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The cellular uptake efficiency of NPs in both cell lines was also evaluated by confocal

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laser scanning microscope (CLSM) (Leica, Heidelberg, Germany). Typically, PC-3 cells or

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MDA-MB-231 cells were seeded on glass-bottom dishes at a density of 2 × 105 cells/well and

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allowed to attach overnight. Then, the cells were exposed to free COU, COU-loaded HATS

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and tLPTS/HATS NPs for 2 h under 5% CO2 at 37 °C. The final COU concentration was 0.5

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µg/mL. After incubation for 2 h, the cells were rinsed with ice-cold PBS twice and fixed with

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4% (v/v) formaldehyde for 15 min at 37 °C. The cell nuclei were stained with Hoechst 33258

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(5 µg/mL). The cells fluorescence was visualized by CLSM.

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2.7. Intracellular trafficking behavior of NPs

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A confocal fluorescent microscope was used to track the intracellular trafficking and

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distribution of NPs. Typically, PC-3 cells were seeded on glass-bottom dishes at a density of

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2 × 105 cells/well and incubated for 24 h. Then, the cells were exposed to COU-loaded HATS

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and tLPTS/HATS NPs for 20 min or 60 min, respectively under 5% CO2 at 37 °C. The final 11

ACCEPTED MANUSCRIPT COU concentration was 0.5 µg/mL. Then, the cells were rinsed with ice-cold PBS three times,

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and the cells lysosomes were labeled by Lyso-Tracker Red (250 nM) for 30 min at 37 °C.

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After another three rinses with ice-cold PBS, the cell nuclei were stained with Hoechst 33258

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(5 µg/mL). The cells were imaged by a CLSM. The Lyso-Tracker Red, COU and Hoechst

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33258 were excited using 561 nm, 488 nm and 345 nm lasers, respectively.

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2.8. Wound healing, cell invasion and migration inhibition assay

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For wound healing assay, PC-3 or MDA-MB-231 cells were seeded in 12-well culture

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plates at 2 × 105 cells/well, and allowed to grow with approximate 90% confluence after 24 h.

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The confluent cell monolayers were wounded with a 10-µL pipette tip. After washed twice

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with PBS, the cells were incubated for 24 h with blank medium (as control), free DTX,

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DTX-loaded HATS NPs and tLPTS/HATS NPs at a concentration of 0.5 µg DTX/mL, as

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well as the corresponding blank tLPTS/HATS NPs under 5% CO2 at 37 °C, respectively.

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Reparation of the wounding area was monitored under a microscope (Olympus, Japan).

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For cell migration assay, PC-3 or MDA-MB-231 cells were seeded in the top transwell

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chambers (Corning, USA) at a density of 3 × 104 cells/well in 100 µL of serum-free medium.

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The lower chambers were filled with 500 µL of culture medium containing 10% FBS as a

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chemo-attractant. Then, the cells were incubated for 24 h with blank medium (as control),

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free DTX, DTX-loaded HATS NPs and tLPTS/HATS NPs at a concentration of 0.5 µg

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DTX/mL, as well as the corresponding blank tLPTS/HATS NPs under standard culture

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conditions, respectively. After that, the PC-3 or MDA-MB-231 cells on the upper surface of

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the membrane (non-invasive cells) were wiped with cotton swabs. The cells on the bottom

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side of the chamber (invasive cells) were fixed by cold 70% ethanol and stained with crystal

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violet, and viewed under a microscope (Olympus, Japan).

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Cell invasion assay was carried out with 1 × 105 cells/well similar to cell migration assay 12

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described above in the transwell chambers coated with matrigel layer.

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2.9. Cell apoptosis assay in vitro Cell apoptosis was determined by Annexin V-FITC apoptosis detection kit according to the

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manufacturer’s protocol. Briefly, PC-3 cells or MDA-MB-231 cells were seeded in 6-well

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culture plates at a density of 4 × 105 cells/well and allowed to attach overnight. Then, the

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cells were treated for 24 h with blank medium (as control), Taxotere®, DTX-loaded HATS

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NPs and tLPTS/HATS NPs, respectively under 5% CO2 at 37 °C. The final concentration of

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DTX was 5 µg/mL. After that, the cells were harvested and suspended in the provided

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binding buffer. Then, 5 µL of Annexin V-FITC was added to the cell suspensions. The

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mixture was incubated for another 15 min at room temperature in dark, followed by adding 5

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µL of PI (propidium iodide). The double-stained cells were immediately analyzed by

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FACScan flow cytometry with 1 × 104 events counted for each sample.

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2.10. Penetration and inhibition on three-dimensional tumor spheroids The hanging drop method was used to prepare the multicellular tumor spheroids of PC-3 or

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MDA-MB-231 cells as described previously [39-41]. Briefly, each well of 48-well culture

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plate was coated with 200 µL of the sterilized agarose solution (2%, w/v) at 80 °C. After

19

being cooled down, each well was added with 900 µL of the cell culture medium. Then, 20

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µL of cell suspension (1 × 103 cells) was suspended on the lid of 48-well culture plate for

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sufficient cell aggregation at 72 h. The resulting cellular aggregates were transferred to the

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bottom of the well, and allowed to grow for another 2 days. Afterwards, the PC-3 and

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MDA-MB-231 tumor spheroids were incubated for 5 days with PBS, Taxotere®, DTX-loaded

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HATS NPs and tLPTS/HATS NPs at a concentration of 10 µg DTX/mL, respectively. The

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spheroid volume was monitored using an inverted phase microscope (Chongqing Optical &

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ACCEPTED MANUSCRIPT Electrical Instrument, Co., Ltd. Chongqing, China). The major (dmax) and minor (dmin)

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diameters of each tumor spheroid were recorded, and the volume was calculated with the

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formula: V = 0.5 × dmax × dmin2. The tumor spheroid volume change ratio was calculated as

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followed: R = (Vi/V0) × 100%, where Vi is the tumor spheroid volume after treatment and V0

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is the tumor spheroid volume prior to treatment.

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To evaluate the penetration ability, the PC-3 and MDA-MB-231 tumor spheroids were

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incubated for 8 h with free COU, COU-loaded HATS and tLPTS/HATS NPs, respectively.

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The final COU concentration was 0.5 µg/mL. After being rinsed with cold PBS twice, the

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tumor spheroids were transferred to a chambered coverslip and examined by CLSM. The

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scanning of tumor spheroid began from the top to the equatorial plane to obtain the Z-stack

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images. Each scanning layer was 8 µm in thickness, and the total scan was 64 µm in depth.

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2.11. Tumor targeting properties of NPs via in vivo imaging

The in vivo biodistribution and tumor targeting capability of NPs were investigated using a

15

near-infrared fluorescent (NIRF) probe of DiR. The male BALB/c nude mice were used to

16

prepare the tumor xenograft model. Briefly, 1 × 107 PC-3 cells in 200 µL of blank medium

17

were subcutaneously inoculated into the right armpit of mice. When the tumor volumes

18

reached 400−500 mm3, 200 µL of various DiR formulations (saline, free DiR, DiR-labeled

19

HATS NPs and tLPTS/HATS NPs) were intravenously injected into the tail vein of mice,

20

respectively. The concentration of DiR was 10 µg/mL. At designated time intervals (1, 3, 6,

21

12, 24 and 48 h after injection), the fluorescent images of mice were acquired by an in vivo

22

molecular imaging system (Carestream; Rochester, NY, USA) with the excitation and

23

emission wavelength at 720 nm and 790 nm, respectively. The X-ray images were also taken

24

for mice location and overlaid with the corresponding fluorescent images. After 48 h, the

25

mice were sacrificed, and the major organs (tumor, heart, liver, spleen, lung and kidney) were

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excised. The ex vivo fluorescent images of the major organs were also detected with the same

2

imaging system as described above.

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2.12. In vivo therapeutic efficacy and safety evaluation The in vivo antitumor efficacy and safety profiles of NPs were evaluated using the PC-3

6

xenograft model. Briefly, 1 × 107 PC-3 cells in 200 µL of blank medium were subcutaneously

7

injected in the right armpit of the male BALB/c nude mice. When the tumor sizes reached

8

around 100 mm3 at day 14 after injection, the mice were randomly divided into four groups

9

(n = 6). Then, the mice were intravenously administrated with saline (as control), and a dose

10

of 10 mg DTX/kg with Taxotere®, DTX-loaded HATS NPs and tLPTS/HATS NPs,

11

respectively at two-day interval for four times. During the experimental period, the tumor

12

volumes were carefully recorded and calculated according to the following formula: V = (a ×

13

b 2)/2, where a and b equaled the length and the width of the tumor, respectively. The body

14

weights of mice were also monitored at 1 day interval. In addition, at day 2 after the fourth

15

injection, 20 µL of blood from the retro-orbital sinus of mice was collected and analyzed by

16

the routine blood test to estimate the blood toxicity of various DTX formulations. At the end

17

of experiment, the mice were sacrificed, and the tumors were excised, photographed and

18

weighed. The net weight variations of the mice were calculated as the body weight at the

19

conclusion of the experiment minus the tumor weight and the body weight at initial day.

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2.13. Statistical Analysis

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All the values are expressed as means ± standard deviation (SD) and statistically evaluated

23

using a one-way analysis of variance (ANOVA). A P-value less than 0.05 were considered to

24

be statistically significant, and a P-value less than 0.01 were considered as highly significant.

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ACCEPTED MANUSCRIPT 1

3. Results and discussion

2

3.1. Synthesis of tLPTS and HATS conjugates Fig. 1A illustrated the synthesis of the amphiphilic targeting molecule of tLPTS. Briefly,

4

the hydrophobic moiety of TOS was coupled to the amine group of Mal-PEG-NH2 by an

5

EDC reaction. Then at mild alkaline condition, the targeting moiety of tLyP-1 peptide was

6

conjugated to the intermediate Mal-PEG-TOS through sulfydryl-maleimide coupling reaction,

7

resulting in formation of tLPTS conjugate.

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ACCEPTED MANUSCRIPT Fig. 1 Synthetic scheme of tLyP-1-PEG-TOS (tLPTS) (A) and HATS (B) amphiphilic

2

conjugates. (C) The 1H-NMR spectra of tLyP-1 (a), Mal-PEG-NH2 (b), the intermediate of

3

Mal-PEG-TOS (c), and the final product of tLyP-1-PEG-TOS (tLPTS) (d) dissolved in D2O.

4

The same colored bars represent the protons (or the carbons attached to the protons) for

5

identifying each compound as follows: yellow, alkyl groups of TOS; brown, tLyP-1 peptide;

6

green, polyethylene glycol; blue, maleimide group. (D) The 1H-NMR spectra of HA (e) and

7

HATS conjugate (f) dissolved in D2O. The yellow and blue bars represent the peaks for

8

succinate ethylene group of TOS and for the N-acetyl group of HA, respectively. (E)

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MALDI-TOF MS spectra of the intermediate Mal-PEG-TOS. (F) MALDI-TOF MS spectra

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of the final tLyP-1-PEG-TOS (tLPTS).

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The structures of the intermediate Mal-PEG-TOS and the final tLPTS conjugate were

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confirmed by 1H NMR spectra. As compared to the spectra of Mal-PEG-NH2 (Fig. 1C (b)),

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the peaks at 3.52−3.64 ppm and 0.68−1.85 ppm belonged to the typical protons of PEG and

15

TOS, respectively in the spectra of Mal-PEG-TOS (Fig. 1C (c)), indicating the intermediate

16

Mal-PEG-TOS was obtained. As shown in Fig. 1C (d), those peaks at 2.62−3.14 ppm and

17

3.86−4.35 ppm for the targeting peptides of tLyP-1 (Fig. 1C (a)) appeared, and meanwhile the

18

characteristic peak at 6.74−6.78 ppm for the maleimide group of the intermediate

19

Mal-PEG-TOS disappeared, which indicated that the final tLPTS conjugate was successfully

20

yielded. The molecular shifts for the intermediate Mal-PEG-TOS and the final tLPTS

21

conjugate was also analyzed by MALDI-TOF as shown in Fig. 1E and 1F, respectively,

22

further confirming the successful synthesis.

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The amphiphilic HATS conjugate was obtained by the hydrophobic TOS chemically

24

conjugated to soluble HA oligomer (Fig. 1B). The structures of both HA and HATS

25

conjugate were confirmed by 1HNMR spectra (Fig. 1D). The characteristic peaks for 17

ACCEPTED MANUSCRIPT methylene groups of sugar unit of HA (Fig. 1D (e)) appeared at 3.22 −4.55 ppm, and those

2

for N-acetyl group ([3H, –COCH3–]) at 1.85−1.98 ppm. Successful synthesis of HATS

3

conjugate (Fig. 1D (f)) was confirmed by the peaks for the succinate ethylene group of TOS

4

[δ = 2.63 ppm, 2H, –COCH2–; δ = 2.78 ppm, 2H, –CH2CO–]. The degree of substitution of

5

TOS to HATS conjugate was determined by the integration ratio of the peaks for the N-acetyl

6

group of HA and for the succinate ethylene group of TOS in 1HNMR spectra, and it was

7

calculated to be 27.35 % in this work.

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3.2. Preparation and characterization of DTX-loaded HATS and tLPTS/HATS NPs As shown in Table 1, the mean diameter of all the DTX-loaded NPs was about 110−120 nm,

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and the PDI was in a range of 0.22 to 0.26, suggesting an acceptable particle size distribution.

12

All the NPs showed good loading capacities for DTX, and the EE was in a range of 85.9% to

13

93.4%. As the tLPTS modification ratio increased, the particle size of DTX-loaded NPs

14

slightly declined with the EE of DTX slightly increased, indicating that the hydrophobic

15

moieties of tLPTS were embedded into the hydrophobic core of HATS-based NPs to form the

16

hybrid tLPTS/HATS NPs [42]. A typical particle size and distribution by DLS and TEM for

17

the prepared DTX-loaded 25%-tLPTS/HATS NPs were shown in Fig. 2. Particle size serves

18

as an important factor in determining its in vivo biological fate. Particles less than 200 nm are

19

not susceptible to the rapid uptake by the reticuloendothelial system (RES) and are easily

20

extravasated into the tumor site via the leaky tumor vasculature [3, 43]. Thus, the developed

21

NPs with size ranging from 110 to 120 nm were predicted to achieve efficient accumulation

22

at tumor tissue by EPR effect.

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The particle surface charge is another important parameter related to the NPs stability in

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vivo. As presented, all the NPs were slightly negatively charged and the zeta potential values

25

were in a range of -5.18 mV to -6.58 mV, due to the presence of carboxylic groups of HA 18

ACCEPTED MANUSCRIPT 1

oligomer. Thus, the developed NPs may repel each other and prevent the aggregation or the

2

in vivo plasma protein adsorption by the electrostatic repulsion, resulting in good in vivo

3

stability [44]. The cumulative release profile of DTX from NPs in vitro was shown in Fig. 2C. Similar to

5

HATS NPs, the 25%-tLPTS/HATS NPs presented a biphases release pattern, that is, the initial

6

burst release for about 12 h and subsequent sustained release.

7

Zeta Potential (mV)

Encapsulation efficiency (%)

Drug content (%)

0.263 ± 0.006

-6.58 ± 0.47

85.94 ± 1.05

7.91 ± 0.09

10%-tLPTS/HATS-DTX 112.5 ± 5.7

0.229 ± 0.035

-5.44 ± 0.61

87.64 ± 0.64

7.38 ± 0.05

25%-tLPTS/HATS-DTX 110.0 ± 3.1

0.244 ± 0.030

-5.18 ± 0.57

93.45 ± 1.97

6.96 ± 0.14

Composition HATS-DTX

Polydispersity

120.3 ± 3.1

All values are expressed as the mean ± SD for at least three different preparations.

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Mean diameter (nm)

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Table 1 Characterization of HATS and tLPTS/HATS nanoparticles containing DTX.

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Fig. 2 (A) Particle size distribution for the prepared tLPTS/HATS NPs by dynamic light

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scattering. (B) Transmission electron micrograph for the tLPTS/HATS NPs. Scale bar: 200

14

nm. (C) In vitro release profiles of DTX from nanoparticles. Data are presented as the mean ± 19

ACCEPTED MANUSCRIPT 1

SD (n = 3).

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3.3. Cytotoxicity assays of DTX-loaded NPs Our previous work [26] revealed that the blank HATS-based polymer had certain

5

cytotoxic effect on cancer cells but nontoxic towards normal cells and it can be used as

6

nanocarrier with specificity and biocompatibility [22, 31]. In this investigation, we evaluated

7

the cytotoxicity of various DTX-loaded HATS-based NPs against two invasive human cancer

8

cell lines of PC-3 and MDA-MB-231 overexpressing both NRP-1 and CD44 receptors [16,

9

17, 45], as compared with Taxotere® (a commercial product of DTX) or free DTX at

10

equivalent doses. Fig. 3A showed the survival rates of PC-3 cancer cell line after treatment at

11

a series of DTX concentrations. Both 10%- and 25%-tLPTS/HATS NPs exhibited stronger

12

anti-proliferative activity than the 0%-tLPTS/HATS NPs (i.e. HATS NPs) at high dose of

13

1−5 µg DTX/mL, which indicated that the tLPTS modification promoted the cytotoxicity of

14

the DTX-formulated NPs. In particular, the 25%-tLPTS/HATS NPs demonstrated much

15

better cytotoxic efficacy against PC-3 cells (P < 0.05), as compared to Taxotere® and free

16

DTX solution. In MDA-MB-231 cells (See supplementary information, Fig. S1), as expected,

17

both the tLPTS functionalized tLPTS/HATS NPs, especially the 25%-tLPTS/HATS NPs

18

exerted much stronger growth inhibition effect than the HATS NPs or free DTX solution at

19

all studied DTX doses. In brief, the DTX-loaded 25%-tLPTS/HATS NPs demonstrated

20

significantly enhanced cytotoxicity against both PC-3 and MDA-MB-231 cancer cells. Based

21

on these results, the modification ratio of tLPTS to HATS was set at 25% if no special

22

instructions in this work.

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Fig. 3 (A) PC-3 cells viabilities of after 48 h incubation with DTX-loaded tLPTS/HATS NPs

3

in comparison with the free DTX, Taxotere®, and HATS-DTX NPs at equivalent doses of

4

DTX (n = 6). Notes: a, P < 0.05, versus free DTX; b, P < 0.05 versus Taxotere®; c, P < 0.05

5

versus HATS-DTX NPs. (B) Flow cytometric quantification of intracellular uptake of

6

COU-loaded NPs in PC-3 cells after incubation for 2 h at 37 °C (n = 3). Notes: ***, P <

7

0.001. (C) Confocal microscope images of intracellular distribution of COU-labeled NPs in

8

PC-3 cells. In the competition experiment (B and C), cells were pretreated with excess free

9

HA or tLyP-1 peptide for 1 h, respectively. (D) Confocal microscope images of intracellular

10

trafficking behavior of COU-loaded HATS NPs and tLPTS/HATS NPs in the PC-3 cells after

11

incubation for 20 min and 60 min, respectively. The concentration of COU was 0.5 µg/mL.

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The Cell nuclei were stained with Hoechst 33258 (blue) and lysosomes were stained by

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Lyso-Tracker Red.

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3.4 Intracellular uptake of NPs in PC-3 and MDA-MB-231 cells To evaluate the intracellular uptake characteristics, nanoparticles loaded with COU were

5

analyzed by flow cytometry and confocal laser scanning microscopy (CLSM), respectively.

6

After incubation for 2 h, the tLPTS/HATS NPs demonstrated significantly enhanced

7

intracellular fluorescent intensity in both PC-3 (Fig. 3B) and MDA-MB-231 cells (See

8

supplementary information, Fig. S2), which were approximately 2.12-fold (P < 0.001) and

9

2.36-fold (P < 0.01) higher than HATS NPs, respectively. The observations from the CLSM

10

(Fig. 3C) for uptake analysis were consistent well with the results of flow cytometry (Fig.

11

3B).

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To confirm the improved cellular uptake of NPs was related to the high expression level

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of CD44 and NRP-1 receptors on both PC-3 and MDA-MB-231 cells, we performed the

14

receptor competitive inhibition assay by pre-incubation with excess free HA or tLyP-1

15

peptides to saturate tumor cell surface receptors. The mean fluorescent intensities of

16

tLPTS/HATS NPs were significantly decreased for both PC-3 (Fig. 3B and 3C) (P < 0.001)

17

and MDA-MB-231 cells (Fig. S2) (P < 0.05). The results confirmed the roles of functional

18

HA and tLyP-1 peptides in the tLPTS/HATS NPs, indicating that the enhanced intracellular

19

uptake of NPs was largely due to the dual receptor-mediated endocytosis, namely by the

20

specific binding of HA to CD44 receptors, and of tLyP-1 peptides to NRP-1 receptors

21

overexpressed on PC-3 and MDA-MB-231 tumor cell surface. Briefly, the cellular uptake

22

studies demonstrated the tLPTS/HATS NPs as highly efficient drug delivery vehicle at the

23

cellular level, which can be taken up by tumor cells via dual receptor-mediated

24

internalization.

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3.5. Intracellular trafficking behavior of NPs The receptor-mediated endocytic process leads to the delivery of transported cargo to

3

lysosomes. To track the intracellular trafficking behavior, NPs loaded COU were observed by

4

CLSM after incubation with PC-3 cells for 20 min and 60 min, respectively with lysosomes

5

stained by Lyso-tracker Red. As shown in Fig. 3D, the green fluorescence of both NPs (HATS

6

NPs and tLPTS/HATS NPs) was dispersed uniformly in the cellular cytoplasm, and the

7

presence of green fluorescence and overlay of green and red (Lyso-tracker Red) fluorescence

8

increased with time after incubation for 20 min and 60 min, respectively. The results

9

indicated that both NPs were orderly trafficked into the cells lysosomes after

10

receptor-mediated endocytosis, and evenly distributed in the cellular cytoplasm. As compared

11

with HATS NPs, a stronger green fluorescence and a relatively higher degree of overlay

12

(yellow) of green and red fluorescence was found for tLPTS/HATS NPs, suggesting the

13

superior intracellular behavior of tLPTS/HATS NPs, that is, the more efficient cellular uptake

14

of NPs by tumor cells and swift distribution into the cellular cytoplasm.

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3.6. Wound healing, cell invasion and migration assay The wound healing (Fig. 4A), cell invasion (Fig. 4B) and migration (Fig. 4C) assays

18

were conducted to evaluate the inhibition ability of DTX-loaded NPs on cell motility and

19

metastasis. For control groups, PC-3 cells showed good motility and high metastasis [17, 45].

20

All the DTX formulations could tremendously depress the wound healing, invasive and

21

migratory activities of PC-3 cells, while the tLPTS/HATS-DTX NPs led to the most

22

significant inhibitory effects as compared to free DTX and HATS-DTX NPs. Interestingly,

23

the blank tLPTS/HATS NPs also exhibited a visible anti-motile activities, indicating the

24

blank tLPTS/HATS NPs itself had mild inhibitory effects on cell motility. Similar results

25

were also observed in the MDA-MB-231 cells assays (data not shown). Several studies have

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ACCEPTED MANUSCRIPT previously demonstrated that the TOS can prevent tumor cell invasion and metastasis [46, 47].

2

As expected, the blank nanocarriers of tLPTS/HATS NPs containing the conjugated

3

“motican” of TOS were found to exhibit efficient anti-motile, anti-invasive and

4

anti-migratory capability, which may make a synergic action with the delivered anticancer

5

agents to treat invasive cancers.

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Fig. 4 Images of wound edge (A), cells invasion (B) and migration (C) of PC-3 cells after

8

incubation with free DTX, DTX-loaded HATS NPs and tLPTS/HATS NPs, and blank

9

tLPTS/HATS NPs formulations for 24 h, respectively (magnification 10 ×).

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Annexin V-PI staining assay was performed to further characterize the cell apoptosis. As

13

shown in Fig. 5A and 5B, the apoptotic rate of untreated PC-3 cells was 3.19%. After exposed

14

to various DTX formulations, the cell apoptosis was enhanced to 16.74% for Taxotere®,

15

5.61% for HATS NPs and 39.50% for tLPTS/HATS NPs, respectively. It was clear that the

16

tLPTS/HATS NPs demonstrated the significantly improved apoptotic feature on PC-3 cells, 24

ACCEPTED MANUSCRIPT which was 2.36-fold and 7.04-fold higher than Taxotere® and HATS NPs, respectively.

2

Similarly, the apoptotic rate of MDA-MB-231 cells (See supplementary information, Fig. S3)

3

under control was 8.52%, and the cell apoptosis was increased to 37.27%, 15.71% and

4

28.38% after treated with Taxotere®, HATS NPs and tLPTS/HATS NPs, respectively. It was

5

demonstrated that the tLPTS/HATS NPs exerted a less apoptotic potency on MDA-MB-231

6

cells than Taxotere®, but displayed 1.81-fold more potency as compared to HATS NPs. The

7

in vitro apoptosis results were in consistency with the cytotoxicity assay.

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3.8. Penetration and inhibitory effects on tumor spheroids

The penetration ability of various NPs into 3D multicellular tumor spheroids was

11

determined by CLSM. After 8 h incubation with free COU, COU-loaded HATS NPs and

12

tLPTS/HATS NPs, confocal microscopic images were taken at different layers of the PC-3

13

tumor spheroid (Fig. 5C) and MDA-MB-231 tumor spheroid (See supplementary information,

14

Fig. S4) from the top to the middle layers. The free COU group displayed the weakest

15

fluorescent intensity, indicating the free COU was incompetent to penetrate into the inner of

16

tumor spheroids, while the moderate fluorescence permeation into the PC-3 and

17

MDA-MB-231 tumor spheroids was observed for HATS NPs. In contrast, the tLPTS/HATS

18

NPs group demonstrated remarkably higher fluorescent intensity throughout the tumor

19

spheroids, suggesting the tLyp-1 peptide-functionalized nanoparticles possessed an excellent

20

ability to penetrate deeper into the inner of solid tumor and distribute more extensively.

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The inhibitory effects on the PC-3 tumor spheroids were also assessed by incubation with

22

various DTX formulations. As shown in Fig. 5D, on the 5th day, the tumor spheroid volume

23

ratios for the PBS control were 168.74 ± 11.41%, indicating the vigorous tumor spheroids

24

growth. After treated with Taxotere®, HATS-DTX NPs and tLPTS/HATS-DTX NPs, the

25

spheroid volume ratios of PC-3 cells were 44.34 ± 2.20%, 59.84 ± 5.94% and 28.88 ± 7.28%, 25

ACCEPTED MANUSCRIPT respectively. All the DTX formulations could effectively suppress the tumor spheroids growth,

2

while the tLPTS/HATS NPs produced the most potency as compared with Taxotere® and

3

HATS NPs (P < 0.05). Similar results were also observed in the MDA-MB-231 tumor

4

spheroid assays (Fig. S5). In brief, a combination of significantly improved endocytosis and

5

cell-penetrating action of the functionalized tLPTS/HATS NPs was believed to play a

6

determinant role in the inhibition effects on tumor spheroids. These results provided direct

7

evidence that the tLPTS/HATS NPs may be available to solve the problem of insufficient

8

drug delivery into the inner of solid tumor that often caused chemotherapeutic failure.

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Fig. 5 (A) Apoptosis profiles of PC-3 cells after 24 h incubation with control (a1), clinic

11

Taxotere® (a2), HATS NPs (a3) and tLPTS/HATS NPs (a4) at equivalent dose of 5 µg

12

DTX/mL, respectively. (B) Apoptosis indexes (AI) were here defined as early and late

13

apoptotic cells (n = 3). Notes: **, P < 0.01; ***, P < 0.001. (C) Confocal microscope images

14

of 3D multicellular tumor spheroids of PC-3 cells after incubation with free COU, HATS NPs

15

and tLPTS/HATS NPs for 8 h. The scale bar represents 75 mm. (D) Inhibition effects on the

16

PC-3 tumor spheroids growth after incubation with various DTX-loaded formulations (n = 3).

17

Notes: a, P < 0.05 versus PBS; b, P < 0.05 versus Taxotere®; c, P < 0.05 versus HATS NPs.

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3.9. In vivo biodistribution of NPs via NIRF imaging The in vivo biodistribution and tumor-targeting efficiency of these NPs were evaluated

3

by NIRF imaging. After i.v. injection of various DiR-labeled formulations via the tail vein,

4

the nude mice bearing PC-3 tumors were then monitored over time (Fig. 6A). In free DiR

5

group, no fluorescence was detected in tumor tissue but a fraction in the liver during the

6

entire imaging. A noticeable signal was observed in tumor tissue as early as 3 h after

7

injection of both NPs (HATS NPs and tLPTS/HATS NPs). The fluorescence signal for both

8

NPs peaked at 24 h and still remained clearly visible at 48 h. In particular, the fluorescence

9

signal of the tLPTS/HATS NPs was widely distributed throughout the whole body of mice at

10

first 1 h, and thereafter preferentially accumulated and intensified in the tumor region until 48

11

h. During the entire imaging, the tLPTS/HATS NPs exhibited much higher and much more

12

lasting accumulation in tumor tissue than the HATS NPs. The hydrophilic HA and PEG shell

13

of the tLPTS/HATS NPs allowed improved serum stability to avoid the rapid non-specific

14

elimination by RES organs of liver and spleen, thus prolonged the blood circulation time.

15

Besides, the tLyp-1 functionalization of the tLPTS/HATS NPs also contributed to tumor

16

tissue-specific recognition and internalization, and extensive penetration into tumor region

17

beyond EPR effect. As shown in Fig. 6B, the ex vivo fluorescent image of excised tumors

18

further confirmed the results observed in vivo. In semi-quantitative analyses (Fig. 6C), the

19

tLPTS/HATS NPs displayed over 1.6-fold and 34.9-fold higher intensity in tumor tissue than

20

the HATS NPs and free DiR, respectively. However, a significant portion of both HA-based

21

NPs was also observed in the liver and spleen, due to the cellular uptake by phagocytic cells

22

of RES system and by liver sinusoidal endothelial cells expressing another HA receptor [48,

23

49]. To some extent, as compared to HATS NPs, the minimized NPs accumulation in the

24

liver and spleen and prolonged blood circulation for the tLPTS/HATS NPs may increase the

25

probability of tLPTS/HATS NPs reaching the tumor site. In brief, these results demonstrated

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ACCEPTED MANUSCRIPT that the designed tLPTS/HATS NPs were available for tumor-specific anticancer drug

2

delivery. The superior tumor-targeting efficiency of the tLPTS/HATS NPs were largely

3

contributed by a combination of extended blood circulation by the hydrophilic HA and PEG

4

shielding effect, an EPR effect arising from the leaky tumor vasculature, and the HA and

5

tLyp-1-mediated tumor-specific recognition and internalization of NPs.

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Fig. 6 (A) In vivo near infrared fluorescent imaging of the PC-3 tumor-bearing nude mice

8

after administration with free DiR, DiR-loaded HATS NPs and tLPTS/HATS NPs. (B) Ex

9

vivo fluorescence images of excised tissues including tumor, heart, liver, spleen, lung and

10

kidney at 48 h postinjection. (C) Semi-quantification of the mean fluorescent intensity of ex

11

vivo tissues (n = 3). Notes: ∗∗, P < 0.01; ∗∗∗, P < 0.001.

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3.10. In vivo therapeutic efficacy and safety evaluation The PC-3 xenografted in male BALB/c nude mice were used for in vivo therapeutic 28

ACCEPTED MANUSCRIPT efficacy study. Various formulations of Taxotere®, DTX-loaded HATS NPs and

2

tLPTS/HATS NPs were administered at 10 mg/kg doses of DTX by four i.v. injections on

3

every three days. The changes of tumor volumes and the body weights were monitored at one

4

day interval for 18 days. As shown in Fig. 7A, the tumor growth suppression rates for the

5

treated groups of Taxotere®, DTX-loaded HATS NPs and tLPTS/HATS NPs were calculated

6

to be 56.7%, 49.3% and 73.8%, respectively as compared to the saline control group. As

7

shown in Fig. 7B and Fig. S6, the isolated tumors for the tLPTS/HATS NPs group exhibited

8

a much smaller average weight and size, further verifying the superior in vivo therapeutic

9

efficacy for the DTX-loaded tLPTS/HATS NPs formulations.

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ACCEPTED MANUSCRIPT Fig. 7 (A) Tumor volume changes for the PC-3 tumor-bearing mice during 18-day treatments

2

(n = 6). (B) The excised tumors weighted and photographed from different treatment groups

3

after the completion of in vivo assays (n = 6). (C) Body weight changes for the tumor-bearing

4

mice and (D) the variation in the net body weight at the conclusion of experiment (n = 6). (E)

5

White blood cell (WBC) count and (F) blood platelet (PLT) count at day 2 after the fourth

6

administration (n = 4). Notes: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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In respect to safety evaluation, the body weight changes of mice during 18-day

9

experimental period were monitored (Fig. 7C), and the net body weight variations at the end

10

of experiment were evaluated (Fig. 7D). Compared with the saline control group, a

11

significant loss of the net body weights of mice was observed for both Taxotere® (P = 0.005)

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and HATS NPs (P = 0.014) groups, while the tLPTS/HATS NPs group exhibited no distinct

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loss of the net body weight (P = 0.147), indicating less systemic toxicity and better safety

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profiles. The hematotoxicity was one of the characteristic side-effects in clinical DTX

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chemotherapy [50], and the routine blood test was also conducted to evaluate the safety

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profiles of various DTX formulations. In Fig. 7E, it was demonstrated that only Taxotere®

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caused an apparent decrease in the white blood cell (WBC) count (P < 0.05 vs control), while

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both HATS and tLPTS/HATS NPs groups produced no serious reduction of WBC count as

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compared with control. Besides, more than 30% of blood platelet (PLT) was found to decline

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in the Taxotere® group (Fig. 7F). Other blood routine examination indexes were not shown

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here due to there were no significant differences. The adverse effect of Taxotere® was due to

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the non-specific distribution and the toxicity of the formulations’ solvent system (ethanol and

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nonionic surfactant Tween-80) [7, 51].

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Invasive cancers are intractable in cancer therapy. In contrast with HATS NPs, the

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tLPTS/HATS NPs demonstrated much better antitumor efficacy and lower systemic toxicity 30

ACCEPTED MANUSCRIPT in the PC-3 xenografts mice model. On the whole, the pronounced in vivo therapeutic

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efficacy of the tLPTS/HATS NPs can be ascribed to the superior tumor-targeting efficiency

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with preferential NPs enrichment in tumor tissues, but minor distribution in non-targeted

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tissues, as verified by in vivo NIRF imaging study. The composite hydrophilic shell of HA,

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PEG and tLyP-1 peptides of the tLPTS/HATS NPs allowed much more lasting blood

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circulation to avoid the rapid non-specific elimination by the RES organs [48, 49]. Moreover,

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the penetrating peptides of tLyp-1 functionalization may help the tLPTS/HATS NPs achieve

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highly efficient drug delivery with tumor tissue-selective recognition and internalizaiton, and

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deep penetration into tumor tissue beyond EPR effect. This enhanced tumor cell recognition

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and tumor tissue penetration was largely contributed by the specific binding of the Cend Rule

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motif R/KXXR/K contained in tLyp-1 peptides to the overexpressed NRP-1 receptors on

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PC-3 tumor cell surface [17], thus promoting internalization of NPs by tumor cells and

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extensive drug delivery into the inner of solid tumor.

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4. Conclusion

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Two modularized amphiphilic polymers of tLPTS and HATS were successfully

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synthesized. The tLPTS/HATS nanoparticles composed of tLPTS and HATS for

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tumor-targeted delivery of DTX were prepared and characterized. The tLPTS/HATS NPs

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displayed significantly enhanced intracellular uptake efficiency, anti-invasion ability,

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cytotoxicity and cell apoptosis in vitro as compared to HATS NPs and the market product of

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Taxotere®, owing to dual receptor-mediated endocytosis process. Moreover, the superior

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penetrating ability and inhibitory effect on multicellular tumor spheroids were observed for

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the tLPTS/HATS NPs. In vivo investigation of NPs by NIRF imaging demonstrated the

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tLPTS/HATS NPs possessed much higher tumor-targeting capacity than HATS NPs. The

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pronounced in vivo therapeutic efficacy with minimal systemic toxicity of the tLPTS/HATS

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NPs was finally verified by the PC-3 xenograft model in athymic nude mice. In brief, it is

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convincing that the multifunctional tLPTS/HATS nanoparticle designed here hold great

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potential as highly efficient anticancer drug delivery vehicle for targeted cancer therapy.

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Acknowledgments

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We would like to acknowledge the NSFC (No.81273454 and No.81473156), National

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key Basic Research Program (No.2013CB932501), Beijing NSF (No.7132113), Doctoral

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Foundation of the Ministry of Education (No.20130001110055) for funding of these works.

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ACCEPTED MANUSCRIPT Table 1 Characterization of HATS and tLPTS/HATS nanoparticles containing DTX. Mean diameter (nm)

Encapsulation efficiency (%)

Drug content (%)

120.3 ± 3.1

0.263 ± 0.006

-6.58 ± 0.47

85.94 ± 1.05

7.91 ± 0.09

10%-tLPTS/HATS-DTX 112.5 ± 5.7

0.229 ± 0.035

-5.44 ± 0.61

87.64 ± 0.64

7.38 ± 0.05

25%-tLPTS/HATS-DTX 110.0 ± 3.1

0.244 ± 0.030

-5.18 ± 0.57

Composition HATS-DTX

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Polydispersity

Zeta Potential (mv)

93.45 ± 1.97

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All values are expressed as the mean ± SD for at least three different preparations.

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6.96 ± 0.14

ACCEPTED MANUSCRIPT Figure Caption Scheme 1. (A) Schematic representation of DTX-loaded tLPTS/HATS NPs. (B) Schematic illustration of tumor-targeted delivery strategy for the tLPTS/HATS NPs. After prolonged blood circulation by composite hydrophilic shell of HA and PEG, the tLPTS/HATS NPs passively

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accumulated at tumor site via EPR effect, followed by promoted intracellular delivery and deep penetration into tumor tissues through CD44 and NRP-1 dual receptor-mediated transcytosis.

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Fig. 1. Synthetic scheme of tLyP-1-PEG-TOS (tLPTS) (A) and HATS (B) amphiphilic conjugates. (C) The 1H-NMR spectra of tLyP-1 (a), Mal-PEG-NH2 (b), the intermediate of Mal-PEG-TOS (c),

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and the final product of tLyP-1-PEG-TOS (tLPTS) (d) dissolved in D2O. The same colored bars represent the protons (or the carbons attached to the protons) for identifying each compound as follows: yellow, alkyl groups of TOS; brown, tLyP-1 peptide; green, polyethylene glycol; blue, maleimide group. (D) The 1H-NMR spectra of HA (e) and HATS conjugate (f) dissolved in D2O. The

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yellow and blue bars represent the peaks for succinate ethylene group of TOS and for the N-acetyl group of HA, respectively. (E) MALDI-TOF MS spectra of the intermediate Mal-PEG-TOS. (F)

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MALDI-TOF MS spectra of the final tLyP-1-PEG-TOS (tLPTS).

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Fig. 2. (A) Particle size distribution for the prepared tLPTS/HATS NPs by dynamic light scattering. (B) Transmission electron micrograph for the tLPTS/HATS NPs. Scale bar: 200 nm. (C) In vitro release profiles of DTX from nanoparticles. Data are presented as the mean ± SD (n = 3).

Fig. 3. (A) PC-3 cells viabilities of after 48 h incubation with DTX-loaded tLPTS/HATS NPs in comparison with the free DTX, Taxotere®, and HATS-DTX NPs at equivalent doses of DTX (n = 6). Notes: a, P < 0.05, versus free DTX; b, P < 0.05 versus Taxotere®; c, P < 0.05 versus HATS-DTX NPs. (B) Flow cytometric quantification of intracellular uptake of COU-loaded NPs in PC-3 cells 37

ACCEPTED MANUSCRIPT after incubation for 2 h at 37 °C (n = 3). Notes: ***, P < 0.001. (C) Confocal microscope images of intracellular distribution of COU-labeled NPs in PC-3 cells. In the competition experiment (B and C), cells were pretreated with excess free HA or tLyP-1 peptide for 1 h, respectively. (D) Confocal microscope images of intracellular trafficking behavior of COU-loaded HATS NPs and tLPTS/HATS

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NPs in the PC-3 cells after incubation for 20 min and 60 min, respectively. The concentration of COU was 0.5 µg/mL. The Cell nuclei were stained with Hoechst 33258 (blue) and lysosomes were

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stained by Lyso-Tracker Red.

Fig. 4. Images of wound edge (A), cells invasion (B) and migration (C) of PC-3 cells after incubation

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with free DTX, DTX-loaded HATS NPs and tLPTS/HATS NPs, and blank tLPTS/HATS NPs formulations for 24 h, respectively (magnification 10 ×).

Fig. 5. (A) Apoptosis profiles of PC-3 cells after 24 h incubation with control (a1), clinic Taxotere®

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(a2), HATS NPs (a3) and tLPTS/HATS NPs (a4) at equivalent dose of 5 µg DTX/mL, respectively. (B) Apoptosis indexes (AI) were here defined as early and late apoptotic cells (n = 3). Notes: **, P < 0.01; ***, P < 0.001. (C) Confocal microscope images of 3D multicellular tumor spheroids of PC-3

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cells after incubation with free COU, HATS NPs and tLPTS/HATS NPs for 8 h. The scale bar

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represents 75 mm. (D) Inhibition effects on the PC-3 tumor spheroids growth after incubation with various DTX-loaded formulations (n = 3). Notes: a, P < 0.05 versus PBS; b, P < 0.05 versus Taxotere®; c, P < 0.05 versus HATS NPs.

Fig. 6. (A) In vivo near infrared fluorescent imaging of the PC-3 tumor-bearing nude mice after administration with free DiR, DiR-loaded HATS NPs and tLPTS/HATS NPs. (B) Ex vivo fluorescence images of excised tissues including tumor, heart, liver, spleen, lung and kidney at 48 h postinjection. (C) Semi-quantification of the mean fluorescent intensity of ex vivo tissues (n = 3). 38

ACCEPTED MANUSCRIPT Notes: ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Fig. 7. (A) Tumor volume changes for the PC-3 tumor-bearing mice during 18-day treatments (n = 6). (B) The excised tumors weighted and photographed from different treatment groups after the

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completion of in vivo assays (n = 6). (C) Body weight changes for the tumor-bearing mice and (D) the variation in the net body weight at the conclusion of experiment (n = 6). (E) White blood cell (WBC) count and (F) blood platelet (PLT) count at day 2 after the fourth administration (n = 4).

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Notes: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Supplementary Files

Tumor-specific penetrating peptides-functionalized hyaluronic acid-D-α-tocopheryl

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succinate based nanoparticles for multi-task delivery to invasive cancers

De-Sheng Liang1,2, Hai-Tao Su1, 2, Ai-Ting Wang1,2, Yu-Jie Liu1,2, Xian-Rong Qi1,2,3,* 1

School of Pharmaceutical Sciences, Peking University, 2Beijing Key Laboratory of

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Molecular Pharmaceutics and New Drug Delivery System, 3State Key Laboratory of

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Natural and Biomimetic Drugs, 38 Xueyuan Road, Haidian District, Beijing 100191, PR

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China

Fig. S1 MDA-MB-231 cells viabilities of after 48 h incubation with DTX-loaded tLPTS/HATS NPs in comparison with the free DTX, Taxotere®, and HATS-DTX NPs at equivalent doses of DTX (n = 6). Notes: a, P < 0.05, versus free DTX; b, P < 0.05 versus Taxotere®; c, P < 0.05 versus HATS-DTX NPs.

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Fig. S2. (A) Flow cytometric quantification of intracellular uptake of COU-loaded NPs in

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MDA-MB-231 cells after incubation for 2 h at 37 °C (n = 3). Notes: ∗, P < 0.05; ∗∗, P <

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0.01. (B) Confocal microscope images of intracellular distribution of COU-labeled NPs in MDA-MB-231 cells. In the competition experiment, cells were pretreated with excess free

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HA or tLyP-1 peptide for 1 h, respectively.

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Fig. S3. (A) Apoptosis profiles of MDA-MB-231 cells after 24 h incubation with control

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(b1), clinic Taxotere® (b2), HATS NPs (b3) and tLPTS/HATS NPs (b4) at equivalent dose of 5 µg DTX/mL, respectively. (B) Apoptosis indexes were here defined as early and late apoptotic cells (n = 3). Notes: *, P < 0.05.

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Fig. S4. Confocal microscope images of 3D MDA-MB-231 multicellular tumor spheroids

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after incubation with free COU, HATS NPs and tLPTS/HATS NPs for 8 h. The scale bar

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represents 75 mm.

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Fig. S5. Inhibition effects on the MDA-MB-231 tumor spheroids growth after incubation with various DTX-loaded formulations (n = 3). Notes: a, P < 0.05 versus PBS; b, P < 0.05 versus Taxotere®; c, P < 0.05 versus HATS NPs.

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Fig. S6. The excised tumors from different treatment groups after the completion of in vivo

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assays (n = 6).