Tumor-Targeting Micelles Based on Linear-Dendritic PEG-PTX8 Conjugate for Triple Negative Breast Cancer Therapy

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00430

Most small molecular chemotherapeutics have poor water solubility and unexpected pharmacokinetics and toxicity to normal tissues. A series of nano drug delivery systems have been developed to solve the problems, among which a micelle based on linear-dendritic polymer-drug conjugates (LDPDCs) is a promising strategy to deliver hydrophobic chemotherapeutics due to its small size, fine stability in blood circulation, and high drug loading capacity. In this work we synthesized a novel amphiphilic linear-dendritic PEG-PTX₈ conjugate which can also encapsulate extra free PTX and self-assemble into uniform ultrasmall micelles with a hydrated diameter of 25.50 ± 0.27 nm. To realize efficient drug delivery to tumor sites, a cyclic tumor homing and penetrating peptide iNGR was linked to the PEG-PTX₈ conjugate. The biological evaluation was performed on a human triple negative breast cancer model. PTX accumulation in tumor at 24 h of the TNBC-bearing mice treated with iNGR-PEG-PTX₈/PTX micelles was significantly enhanced (P < 0.001, two-way ANOVA) compared to that of Taxol and untargeted MeO-PEG-PTX₈/PTX micelles. Furthermore, iNGR-PEG-PTX₈/PTX micelles showed an obvious strong antitumor effect, and the median survival time of TNBC bearing mice treated with iNGR-modified micelles was significantly extended compared to Taxol. Therefore, this smart micelle system may be a favorable platform for effective TNBC therapy.

Full text of DOI:10.1021/acs.molpharmaceut.7b00430

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Article
Tumor-Targeting Micelles Based on Linear-Dendritic PEG-
PTX8 Conjugate for Triple Negative Breast Cancer Therapy
Yujie Zhang, Yifei Lu, Yu Zhang, Xi He, Qinjun Chen, Lisha
Liu, Xinli Chen, Chunhui Ruan, Tao Sun, and Chen Jiang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00430 • Publication Date (Web): 23 Aug 2017
Downloaded from http://pubs.acs.org on August 24, 2017
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TumorꢀTargeting Micelles Based on Linearꢀ
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Dendritic PEGꢀPTX Conjugate for Triple
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Negative Breast Cancer Therapy
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Yujie Zhang, Yifei Lu, Yu Zhang, Xi he, Qinjun Chen, Lisha Liu, Xinli Chen,
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Chunhui Ruan, Tao Sun, Chen Jiang
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Key Laboratory of Smart Drug Delivery, Ministry of Education, State Key
Laboratory of Medical Neurobiology, Department of Pharmaceutics, School of
Pharmacy, Fudan University, Shanghai 200032, China
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Corresponding author: Chen Jiang, Tel: +86ꢀ21ꢀ5198ꢀ0079; Fax: +86ꢀ21ꢀ5198ꢀ
079; Eꢀmail: jiangchen@shmu.edu.cn.
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Table of Contents/Abstract Graphic
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KEYWORDS: polymerꢀdrug conjugates, linearꢀdendritic, micelle, triple negative breast cancer,
tumor targeting
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ABSTRACT: Most small molecular chemotherapeutics have poor waterꢀsolubility, unexpected
pharmacokinetics and toxicity to normal tissues. A series of nano drug delivery systems have
been developed to solve the problems, among which micelle based on linearꢀdendritic polymerꢀ
drug conjugates (LDPDCs) is a promising strategy to deliver hydrophobic chemotherapeutics
due to its small size, fine stability in blood circulation and high drug loading capacity. In this
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work we synthesized a novel amphiphilic linearꢀdendritic PEGꢀPTX conjugate which can also
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encapsulate extra free PTX and selfꢀassemble into uniform ultraꢀsmall micelles with a hydrated
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diameter of 25.50±0.27 nm. To realize efficient drug delivery to tumor sites, a cyclic tumor
homing and penetrating peptide iNGR was linked to the PEGꢀPTX conjugate. The biological
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evaluation was performed on a human triple negative breast cancer model. PTX accumulation in
tumor at 24 h of the TNBCꢀbearing mice treated with iNGRꢀPEGꢀPTX /PTX micelles was
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significantly enhanced (P<0.001, twoꢀway ANOVA) compared to that of Taxol and untargeted
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MeOꢀPEGꢀPTX /PTX micelles. Furthermore, iNGRꢀPEGꢀPTX /PTX micelles showed obviously
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strong antitumor effect and the median survival time of TNBC bearing mice treated with iNGRꢀ
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modified micelles was significantly extended compared to Taxol. Therefore, this smart micelle
system may be a favorable platform for effective TNBC therapy.
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INTRODUCTION
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Most small molecular anticancer drugs are poorly waterꢀsoluble, eliminated quickly in vivo,
lacking in tumorꢀtargeting ability and toxic to normal tissues. These disadvantages greatly
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, 2
limited their therapeutic effect and clinical application.
With the development of
nanotechnology, a variety of innovative nano drug delivery systems (NDDS) including
nanoparticles, liposomes and micelles have been explored to increase solubility, improve
pharmacokinetics, enhance tumor targeting, decrease systemic toxicity and eventually achieve
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optimal therapeutic benefits of hydrophobic drugs.
Many liposomal, micellar or particulate nano formulations load hydrophobic drugs through
physical entrapment via hydrophobic interaction. However, the resulting nanoparticles generally
have low drug loading content and the weak intermolecular force may lead to internal instability
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of the nanoparticle structure and burst drug release in physiological medium.
Even the FDA
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approved Abraxane, a nanoparticle formulation of albumin bounded paclitaxel (PTX), was found
unstable in blood circulation, leading to drug leakage, unexpected pharmacokinetic properties,
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and offꢀtarget effect postꢀadministration.
Another strategy for NDDS is to link the hydrophobic therapeutics to macromolecules or
polymers through covalent bonds. The covalent linkage is more stable than the intermolecular
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force in blood circulation and premature drug release can be avoided.
Micelleꢀforming
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polymerꢀdrug conjugates (PDCs) is attracting increasing attention in the nanomedicine field due
to its relatively good stability in blood stream and suitable assembled size (10ꢀ100 nm) to take
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advantage of EPR effect and accumulate in tumor site.
Over the past decades, researchers
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have synthesized various PDCs with different architectures. Traditional linear PDCs usually
linked mono drug molecule and the use of high molecular weight linear polymers often leads to
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low drug loading content.
Additionally, drug conjugation might change the polymer
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composition or amphiphilicity, which could disrupt the nanostructure formation. Graftꢀ
copolymerꢀdrug conjugates can conjugate several drug molecules onto the side chains, but the
grafted copolymer is composed of more than one organic monomers with appropriate
polymerization degree, so the total molecular weight is always high, and the steric hindrance of
the reactive side chains will increase difficulty for drug conjugation, resulting in reduced drug
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loading capacity. Moreover, reagents used in the synthetic process of new polymeric materials
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might be toxic, nonꢀbiocompatible and remain in the materials, causing potential safety problems,
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and techniques employed in the nanostructure formation may have some limits.
In
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comparison, linearꢀdendritic polymerꢀdrug conjugate (LDPDC) is a unique class of PDC with
wellꢀdefined structure, tunable aggregation properties, significantly increased drug loading
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content and superior stability.
Therefore, it is a promising strategy to utilize LDPDC to
delivery drugs for tumor therapy.
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In this work, we aimed to design a novel LDPDC which utilize only one sort of dendritic
polymer with favorable molecular weight to achieve improved drug properties such as longer
blood circulation time, enhanced in vivo stability and targeting ability to tumor sites. PTX, a very
potent chemotherapeutic agent and almost insoluble in water, was used as a model drug. Poly
(ethylene glycol) (PEG) is one of the most widely used synthetic hydrophilic polymers with
favorable biocompatibility and has been utilized to modify various hydrophobic
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chemotherapeutics to increase solubility and prolong blood circulation time.
The commercial
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PEGꢀNH (Mw=5000) was transformed into linearꢀdendritic PEG with eight hydroxyl ends
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(PEGꢀOH ) through three rounds of anhydride acylation and acid hydrolysis, then PTX was
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conjugated to PEGꢀOH through a linker named 4ꢀnitrophenyl chloroformate. With PEG as
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hydrophilic segment and PTX as the hydrophobic segment, the resulting LDPDC PEGꢀPTX8
could selfꢀassembly into stable homogeneous ultraꢀsmall micelles. Additionally, the amphiphilic
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dendritic PEGꢀPTX can also serve as a drug carrier and encapsulate extra hydrophobic drugs
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into the hydrophobic core.
A promising drug delivery system should not only possess high drug loading capacity, good
stability in blood stream but also realize targeted drug delivery into the lesion location. Since
PTX is the firstꢀline chemotherapeutics for the treatment of breast cancer, we intended to
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evaluate the linearꢀdendritic PEGꢀPTX micelles on breast cancer. Triple negative breast cancer
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(TNBC) is the most malignant breast cancer subtype which lacks expression of estrogen receptor
(ER), progesterone receptor (PR) and human epidermal growth factor receptorꢀ2 (HERꢀ2),
accordingly it is considered relatively difficult to try targeted therapy for TNBC. It was reported
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that neuropilinꢀ1 (NRPꢀ1) is overexpressed on tumor vessels and TNBC, which can be
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considered as targets for drug delivery.
A novel designed cyclic peptide iNGR
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(CRNGRGPDC, C&C disulfide bridge) contains a vascular homing motif (NGR) and a CendR
motif (R/KXXR/K) that can penetrate into tumor tissues more effectively than the standard NGR
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peptide. In blood circulation, iNGR specifically and rapidly bound to CD13 overexpressed on
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the surfaces of tumor neovascular endothelial cells. Afterwards, the cyclopeptide suffered a
proteolytic process on the cell surface and exposed the internal CendR motif of iNGR. As a
consequence, the resulting linear iNGRt (CRNGR) could penetrate tumor cells, which is
mediated by NRPꢀ1 (Scheme 1). This strategy allows the activation of the CendR motif only in
the targeted tissue rich in neovasculature, avoiding NRPꢀ1 activation in normal tissues.
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Therefore, iNGR peptide was modified on the linearꢀdendritic PEGꢀPTX conjugates to enhance
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targeting ability to TNBC. The in vitro and in vivo properties and effects of the iNGRꢀPEGꢀPTX8
micelles were fully evaluated in the study.
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Scheme 1. Schematic illustration presenting the preparation of iNGRꢀPEGꢀPTX8/PTX micelles
and the in vivo performance through CD13 and NRPꢀ1 receptors.
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MATERIALS AND METHODS
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Materials
AzideꢀPEG ꢀamine trifluoroacetic acid salt (N ꢀPEG ꢀNH TFA, Mw=5000) and Methoxyꢀ
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5k
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PEG ꢀamine (MeOꢀPEG ꢀNH , Mw=5000) were purchased from Jenkem technology Co., Ltd.
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(Beijing, China). Paclitaxel (PTX) was purchased from Meilune Biological technology Co., Ltd.
(Dalian, China). iNGR and iNGRt peptides with alkynyl modification were synthesized by China
Peptides Co., Ltd. (Suzhou, China). Sodium ascorbate, 4ꢀdimethylaminopyridine (DMAP) and
N,N,N′,N′′,N′′ꢀpentamethyldiethylenetriamine (PMDETA) were purchased from J&K chemical
Co., Ltd. (Shanghai, China). [4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT)
and coumarinꢀ6 were purchased from SigmaꢀAldrich (St. Louis, MO, USA). Benzaldehyde
dimethyl acetal, 2,2ꢀBis (hydroxymethyl) propionic acid, tertꢀbutyl methyl ether (TBME) and 4ꢀ
(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid (HEPES) was purchased from Aladdin
Industrial Corporation (Shanghai, China). Fluorescent dye BODIPY was synthesized according
to the literature before. All other chemicals were purchased from Sinopharm Chemical Reagent
Co., Ltd (Shanghai, China) unless mentioned otherwise.
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Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL
(Carlsbad, CA, USA). DNA Fragmentation Detection Kit and Cell Cycle Detection Kit were
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purchased from KeyGEN BioTECH (Nanjing, China). Dꢀluciferin potassium was purchased
from Pierce (Rockford, USA).
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Human umbilical vein endothelial cells (HUVECs) was obtained from American Type Culture
Collection (ATCC, Rockville, MD, USA). MDAꢀMBꢀ231 cell line was purchased from
Shanghai institutes for cell resource center, Chinese Academy of Sciences (Shanghai, China).
MDAꢀMBꢀ231/luci cell line was purchased from Cell Biolabs, Inc (San Diego, USA). Female
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SD rats and nude mice were supplied by the Animal Center of School of Pharmacy, Fudan
University.
Synthesize and characterization of linear-dendritic PEG-PTX Conjugates.
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MeOꢀPEGꢀOH and N ꢀPEGꢀOH were synthesized according to the synthetic route (Figure 1)
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as described previously. MeOꢀPEGꢀOH and MeOꢀPEGꢀOH were also produced during the
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process. PTX was conjugated to MeOꢀPEGꢀOH (n=2, 4, 8) and N ꢀPEGꢀOH using 4ꢀ
n
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nitrophenyl chloroformate as a linker. In brief, benzylideneꢀ2, 2ꢀbis(oxymethyl) propionic
anhydride (BBOPA) was synthesized through two steps of dehydrationꢀcondensation reaction.
Then MeOꢀPEGꢀNH (Mw=5000) and N ꢀPEGꢀNH (Mw=5000) were transformed into MeOꢀ
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PEGꢀOH and N ꢀPEGꢀOH respectively through three rounds of BBOPA acylation and acid
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hydrolysis. MeOꢀPEGꢀOH (150 mg, 1 equiv.) and excess 4ꢀnitrophenyl chloroformate (387 mg,
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64 equiv.) were dissolved in 5 mL dry dichloromethane (DCM) under the protection of nitrogen,
then pyridine (309 µL, 128 equiv.) was added dropwise. The reaction mixture was stirred at
room temperature for 24 h. After completion, the white precipitation was filtered off and washed
with cold DCM. Then the filtrate was concentrated and purified by column chromatography on
Sephadex LH 20 using methanol as eluent. The solvent was removed under reduced pressure to
get the desired product (a7 in Figure 1). Then PTX (20 equiv.) and DMAP (4 equiv.) were added
to the above product and dissolved in 4 mL of dry DCM under the protection of nitrogen. After
24 h stirring under room temperature, the mixture was also purified on Sephadex LH 20 to get
the target product MeOꢀPEGꢀPTX . N ꢀPEGꢀPTX was synthesized by the same procedures.
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Finally 237 mg MeOꢀPEGꢀPTX and 307.5 mg N ꢀPEGꢀPTX was obtained. By the way, MeOꢀ
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PEGꢀPTX and MeOꢀPEGꢀPTX were also synthesized and purified in the same method.
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Alkynyl iNGR (or iNGRt) peptide was linked to the terminus of N ꢀPEGꢀPTX via copper(I)ꢀ
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catalyzed azideꢀalkyne cycloaddition (CuAAC) click reaction. N ꢀPEG ꢀPTX (85 mg, 1 equiv.)
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and excess alkynylated iNGR (27 mg, 3 equiv.) were dissolved in 3 mL of dry
dimethylformamide, then sodium ascorbate (15 mg, 9 equiv.), PMDETA (5.4 µL, 3 equiv.) and
solid CuI (4.8 mg, 3 equiv.) were added to the solution in order. The whole process was carried
out under the protection of Argon. The reactant was stirred for 12 h at 40 . After completion,
The mixture was dialyzed against PBS buffer containing 10 mM EDTAꢀ2Na (pH=7.4) for 12 h,
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deionized water for 24 h, followed by freezeꢀdrying. Finally, 90 mg iNGRꢀPEGꢀPTX and 30 mg
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iNGRtꢀPEGꢀPTX were obtained.
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Figure 1. Synthetic route of MeOꢀPEGꢀPTX , iNGRꢀPEGꢀPTX and iNGRtꢀPEGꢀPTX .
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Products of each step were characterized by NMR spectrometer (400 MHz, Bruker, Billerica,
MA), and iNGRꢀPEGꢀPTX and iNGRtꢀPEGꢀPTX were characterized by IR spectrum
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additionally.
Preparation and characterization of micelles based on different PEG-PTX conjugates.
To optimize the micelle formulation, a series of micelles composed of different linearꢀdendritic
PEGꢀPTX conjugates and encapsulated PTX amount were prepared via dialysis method. In
detail, a certain amount of PTX was weighed and dissolved in methanol to concentration of 1
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mg/mL and all of the PEGꢀPTX conjugates were dissolved in methanol to final concentration of
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5 mg/mL separately. 2 mg of each kind of PEGꢀPTX conjugates and 5%, 10%, 20% and 40% （
w/w ） free PTX were mixed up and vortex for 30 s and then sealed in a dialysis bag
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(diameter=18 mm, MWCO=5000) followed by dialysis against deionized water for 12 h. Then
the liquid in dialysis bag was collected and filtered through a 0.22 µm filter to get the final
micelle solution. Size and PDI of the freshly prepared micelles were measured by dynamic light
scattering (DLS) (Zetasizer NanoꢀZS, Malvern, U.K.) and reꢀmeasured at day 7. The
morphological images of the PTX loaded micelles were photographed by transmission electron
microscope (TEM, Tecnai G2 spirit Biotwin, FEI).
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PTX loading capacity of the micelles was determined by HPLC through alkaline lysis method.
Briefly, 10 µL of the micelles was diluted 10 times with distilled water and then treated with 100
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µL of 2 N NaOH aqueous solution for 30 min at 37 °C water bath, afterwards, 200 µL of 1 N
HCl aqueous solution was added to neutralize the excess alkaline. The salt produced in this step
was precipitated by mixing with acetonitrile (ACN) and centrifuged at 12000 rpm for 10 min for
three times. PTX standard sample was also treated with the same method to obtain the standard
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curve. 20 µL of the supernatant was analyzed by HPLC at 227 nm with 40% ACN and 60% H O
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as mobile phase.
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In vitro PTX release studies.
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In vitro PTX release from the micelles was performed using dialysis method (n=3). In brief,
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0.3 mL solution of free PTX (dissolved in methanol), Taxol, MeOꢀPEGꢀPTX /PTX and iNGRꢀ
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PEGꢀPTX /PTX micelles with an equal PTX concentration (0.5 mg/mL) was sealed into a
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dialysis bag (MWCO=3500) and immediately placed into 12 mL dialysis medium (pH=7.4 PBS,
containing 1N sodium salicylate). The experiment was carried out at 100 rpm, 37 °C on the
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Shaking Bath for 72 h. 0.3 mL of the medium was withdrawn and equal volume of fresh release
medium was replenished at various time points. PTX concentration of each sample was
determined by HPLC at 227 nm.
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Cell culture
MDAꢀMBꢀ231 cells and MDAꢀMBꢀ231/luci cells were grown in DMEM containing 10 mM
HEPES and 10% FBS at 37 °C with 5% CO . HUVEC cells were cultured in DMEM added with
2
10% FBS and 1% v/v penicillinꢀstreptomycin solution at 37 °C with 5% CO2.
Cell uptake studies.
MDAꢀMBꢀ231 cells and HUVECs were seeded in 24ꢀwell plates (CorningꢀCoaster, Tokyo,
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Japan) at a density of 8×10 cells per well. When achieving 80ꢀ90% confluence, the cells were
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incubated with nonꢀtargeting micelles (MeOꢀPEGꢀPTX micelles), iNGRꢀmodified micelles
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(with 10%, 20% and 40% iNGR modification) and iNGRtꢀmodified micelles (with 20% iNGRt
modification, determined by the cellular uptake results of iNGRꢀmodified micelles) at the
concentration of 20 µg/mL PEGꢀPTX conjugates in serumꢀfree DMEM. All the micelles were
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encapsulating with 0.5% (w/w) coumarinꢀ6 as a fluorescent probe through dialysis method as
described above. After 30 min incubation, cells were rinsed with PBS for three times and then
0.3 mL of Hank’s was added to each well. The cellular uptake of micelles was visualized and
photographed by inverted fluorescent microscope (Leica Wetzlar).
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In vitro anticancer efficacy study.
In vitro anticancer efficacy of the micelles was evaluated by MTT assay (n=4). MDAꢀMBꢀ231
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cells were seeded at a density of 5 × 10 cells/well in 96ꢀwell plates. After 24 h incubation, cells
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were then washed for three times with PBS and exposed to 200 µL of various concentrations of
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Taxol, MeO−PEG−PTX /PTX, 20% iNGR−PEG−PTX /PTX micelles (mentioned as iNGRꢀ
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PEGꢀPTX/PTX below) and 20% iNGRt−PEG−PTX /PTX micelles (mentioned as iNGRtꢀPEGꢀ
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PTX/PTX below) at 37 °C for 48 h. Then the medium was removed and cells were washed with
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PBS for three times and 150 µL of MTT (0.5 mg/mL) solution was added into each well and
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further incubated at 37 °C for 4 h. Next, the solution was sucked up and 200 µL of DMSO was
added to each well to dissolve the formazan crystals formed by the living cells. Then the 96ꢀwell
plates were balanced on the air bath for 10 min at 37 °C in the dark. Cells without treatment
served as control. Absorbance was read at 570 nm by dual wavelength detection using a
Multiskan MK3 microplate reader (Thermo Scientific, Waltham, Massachusetts, USA). Cell
viability was calculated as the survival percentage of control.
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Cell cycle analysis
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MDAꢀMBꢀ231 cells were seeded on a sixꢀwell plate and cultured at 37 °C for 24 h. When 80ꢀ
90% confluence was reached, cells in each well were treated with different formulations of PTX
at an equal concentration of PTX (1 µg/mL) in FBSꢀfree medium. After a 30ꢀmin incubation, the
medium was removed and cells were washed with Hank’s for three times and cultured with fresh
complete medium for another 24 h. Next, Cells were collected and centrifuged at 2000 rpm, then
washed with cold PBS and fixed with 500 µL cold 70% ethanol overnight. Then the fixed cell
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suspension was centrifuged and washed with PBS redispersed in 300 µL PBS. Cells were treated
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with RNase A for 30 min at 37 °C and then with 80 µL PI for another 30 min. Fluorescence was
measured by a flow cytometer (FACS, BD Biosciences, Bedford, MA) and histograms of cell
number versus PI intensity were used to determine the percentage of cells in each phase of the
cell cycle.
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Modeling of triple negative breast cancer.
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Female BALB/C nude mice of ~20 g body weight were xenografted with 1×10 MDAꢀMBꢀ
231/luci cells which were suspended in 100 µL Matrigel solution (5 mg/mL in PBS) by in situ
injection into the second right mammary fat pad of nude mice and were raised under SPF
laboratory conditions.
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In vivo imaging.
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AzaꢀBODIPY (Ex=700 nm, Em=730 nm) was encapsulated as a NIR probe in the nonꢀ
targeting and iNGRꢀmodified micelles at a final concentration of 20 µg/mL. Micelles were
injected intravenously via tail vein into MDAꢀMBꢀ231 bearing mice at concentration of 0.2 mg
BODIPY/kg. The in vivo biodistrabution of micelles at different time was traced and visualized
by Xenogen IVIS Spectrum with Living Image Software 4.2 (Caliper Life Science, Hopkinton,
Massachusetts, USA). Mice were sacrificed at 24 h and tumors and organs was collected ex vivo
and also photographed by IVIS.
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Pharmacokinetics and Biodistribution studies.
To investigate in vivo pharmacokinetics (PK) of the MeOꢀPEGꢀPTX /PTX and iNGRꢀPEGꢀ
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PTX /PTX micelles, healthy female SD rats were fasting for 18 h before experiment. The rats
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were randomly divided into three groups and then administrated with Taxol, MeOꢀPEGꢀPTX8
/PTX and iNGRꢀPEGꢀPTX /PTX micelles at a dose of 10 mg PTX/kg via caudal vein injection
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(n=6) respectively. 0.5 mL blood was collected at each time point and centrifuged at 3500 rpm
for 10 min to get supernatant for further treatment. Diazepam was used as an internal standard
for determination of PTX concentration. All the plasma samples were treated by the previous
method and analyzed by HPLC.
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Biodistribution of PTX in MDAꢀMBꢀ231 bearing nude mice was performed when tumor
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volume reached approximately 500 mm (n=4). Taxol and the micelles were also injected via tail
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vein at the same dose of PK test. Mice were sacrificed at 2 h and 24 h separately. Tumors and
normal organs (including heart, liver, spleen, lung and kidney) were taken out, washed with cold
saline, dried over filter paper, weighed and stored at ꢀ20 °C before analysis. Docetaxel (DTX)
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4
was employed as an internal standard. The samples was prepared as described and PTX
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concentration was measured by LC/MS/MS (AB 4000 Q TRAP). ZORBAX 300 ExtendꢀC18
column (2.1×150 mm, 3.5 µm) was employed and flow rate was 0.2 mL/min. the mobile phase
was 80% methanol and 20% water containing 0.1% formic acid. The retention time of PTX and
DTX was 2.18 min and 2.26 min separately.
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Antitumor efficacy
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Nude mice with MDAꢀMBꢀ231/luci xenograft were randomly divided into four groups (n=6)
according to the bioluminescence signal scanned by the in vivo realꢀtime bioluminescence
system which reflects the tumor size of the mice. The mice were administrated with saline,
Taxol, MeO−PEG −PTX /PTX and iNGR−PEG −PTX /PTX micelles (at an equal dose of 10
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k
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5k
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mg/kg PTX) at day 15 post implantation. Weight and tumor volume were measured every other
day.
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Terminal deoxynucleotidyl transferase dUTP nickꢀend labeling (TUNEL) assay was performed
on frozen tumor slices from TNBC bearing mice administrated with different PTX formulations
using a DNA fragmentation detection kit according to the instruction. Pictures of the stained
tumor slices were photographed under the inverted fluorescent microscope.
RESULTS AND DISCUSSION
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Synthesize and characterization of linear-dendritic PEG-PTX Conjugates
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Compound BBOPA, polymer a1ꢀa7, b1ꢀb7, MeOꢀPEGꢀPTX and MeOꢀPEGꢀPTX were all
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successfully synthesized and characterized by H NMR spectra (Supporting Information, Figure
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S1ꢀS18). As shown in Figure 2A and 2B, peaks between 7 and 8 ppm represent the protons of
aryl ring of PTX and peaks at 3.5ꢀ3.6 ppm represent the protons of repeated ethylene oxide unit
of PEG, which indicate the successful synthesis of MeOꢀPEGꢀPTX and N ꢀPEGꢀPTX .
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Calculated by H NMR spectrum, 6.67 and 6.56 PTX was conjugated to MeOꢀPEGꢀOH and N ꢀ
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PEGꢀOH per molecule on average, respectively. iNGRꢀPEGꢀPTX and iNGRtꢀPEGꢀPTX8 were
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characterized by IR spectra additionally. The azide peak of N ꢀPEGꢀPTX showed on IR
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ꢀ1
spectrum was ~2109 cm , and the peak was disappeared on the IR spectrum of iNGRꢀPEGꢀ
PTX (Figure 2C) and iNGRtꢀPEGꢀPTX (Figure 2D), suggesting that the desired conjugates
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were synthesized successfully.
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Figure 2. Characterization of different PEGꢀPTX conjugates. (A) H NMR spectrum of MeOꢀ
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PEGꢀPTX ; (B) H NMR spectrum of N ꢀPEGꢀPTX ; (C) IR spectrum of iNGRꢀPEGꢀPTX and
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(D) IR spectrum of iNGRtꢀPEGꢀPTX8.
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Preparation and characterization of micelles based on different PEG-PTX conjugates.
Since the linearꢀdendritic PEGꢀPTX (n=2, 4, 8) conjugates are amphiphilic, we assumed that
n
they could also serve as carriers to load free hydrophobic drugs in the hydrophobic core.
Therefore, we prepared a series of micelles with different amount of free PTX to get the optimal
formulation of the PEGꢀPTX based micelles. Size distribution and PDI of micelles based on
different PEGꢀPTX conjugates was concluded in Figure 3A and 3B, separately. We found that
1
micelle structures formed spontaneously upon dialysis against deionized H O. By comparison in
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size, PDI and dynamic stability, the micelle of PEGꢀPTX encapsulated 5% free PTX was
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considered as the optimal formulation with good fine in vitro stability, low PDI and ultraꢀsmall
size (Table 1). When incubated with 10% FBS at 37 for 12 h or diluted 10 and 100 times with
PBS (pH=7.4) buffer, the micelles size showed no significant change (Supporting Information,
Figure S19 and Figure S20), which proved that it was considerably stable against serum and
dilution in physiological buffers.
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Figure 3. Characterization of various micelles. (A) PDI and (B) size distribution of different
micelles based on PEGꢀPTX conjugates (n=3); Size distribution by DLS of (C) MeOꢀPEGꢀ
PTX8/PTX micelles and (D) iNGRꢀPEGꢀPTX8/PTX micelles (n=3); and representative TEM
images of (E) MeOꢀPEGꢀPTX8/PTX micelles and (F) iNGRꢀPEGꢀPTX8/PTX micelles.
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TEM images show that the morphology of MeOꢀPEGꢀPTX /PTX and iNGRꢀPEGꢀPTX /PTX
8 8
micelles is coreꢀshelled and homogeneous (Figure 3E and 3F) with diameter of 19.81 ± 0.47 nm
and 21.47 ± 0.47 nm respectively, which are slightly smaller than the hydrated diameters
measured by DLS.
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Through HPLC determination, drug loading capacity of MeOꢀPEGꢀPTX /PTX and iNGRꢀ
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PEGꢀPTX /PTX micelles was 54.70 ± 1.41% and 52.26 ± 0.77%, respectively, higher than most
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PTXꢀloaded nanoparticles and micelles reported before. The overall properties of the PTX
loaded micelles were summarized in Table 1.
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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5
5
5
5
5
5
5
5
5
6
Table 1. Summary of micelle properties (Data are presented as mean ± SD, n=3).
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Formulation
Size (ZꢀAverage, PDI
d.nm)
Drug
Capacity (%)
Loading
MeOꢀPEGꢀ
25.50 ± 0.27
26.17 ± 0.13
29.06 ± 0.31
0.082 ± 0.002
54.70 ± 1.41
PTX /PTX
8
iNGRꢀPEGꢀ
0.126 ± 0.014
0.142 ± 0.022
52.26 ± 0.77
53.17 ± 0.62
PTX /PTX
8
iNGRtꢀPEGꢀ
PTX /PTX
8
4
5
6
7
8
9
0
1
2
In vitro PTX release studies.
The in vitro drug release properties under physiological condition was simulated by dialysis
method in PBS (pH=7.4). As shown in Figure 4, free PTX was rapidly released from the dialysis
bag (MWCO=3500) while PTX release rate from Taxol was relatively slow. This is because the
solvent of Taxol, Cremophor EL, is an amphiphilic polymer as well as a surfactant which can
form micelles and encapsulate PTX in the core by selfꢀassembling upon countering with water.
Both of the MeOꢀPEGꢀPTX /PTX and iNGRꢀPEGꢀPTX /PTX micelles only release ~10% of
1
1
1
8
8
total PTX until 72 h, which indicated that the covalent linkage between PEG and PTX were
relatively stable under physiological conditions and burst release of the loaded drug was avoided
to some extent. However, the sufficient enzymes in the tumor cells permit an accelerated
13
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hydrolysis of the carbonate ester to release PTX.
The drug biodistribution study (see below)
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also proved that prototype PTX was released from the micelles in the tissues.
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Figure 4. In vitro drug release behaviors from different PTX formulations in PBS (pH 7.4) at 37
°C with free PTX (dissolved in methanol) as the control group. Data are represented as mean ±
SD (n = 3).
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Cellular uptake of the micelles.
HUVEC is widely used as a model to mimic tumor neovascular cells and it was reported that
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CD13, NRPꢀ1/2, and Matriptase are overexpressed on HUVECs.
Human MDAꢀMBꢀ231 cell
line overexpresses NRPꢀ1 and was chosen to mimic TNBC. The cellular uptake efficiency of the
micelles was evaluated in HUVECs and MDAꢀMBꢀ231 cells. Firstly micelles with different
percent of iNGRꢀPEGꢀPTX (w/w) at a concentration of 20 ꢁg/mL PEGꢀPTX conjugates were
1
1
1
1
8
8
coꢀincubated with HUVECs and 20% iNGRꢀPEGꢀPTX /PTX micelles showed enough targeting
8
to HUVECs (Supporting Information, Figure S21). So we selected 20% iNGRꢀmodified micelles
and 20% iNGRtꢀmodified micelles to do subsequent experiments. Images under fluorescence
14
microscope suggested that HUVECs treated with iNGRꢀPEGꢀPTX /Cou6 and iNGRtꢀPEGꢀ
8
1
5
PTX /Cou6 micelles exhibited obviously enhanced internalization compared to nonꢀtargeting
8
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MeOꢀPEGꢀPTX /Cou6 micelles (Figure 5). MDAꢀMBꢀ231 cells treated with iNGRtꢀPEGꢀ
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PTX /Cou6 showed significantly higher uptake efficiency than nonꢀmodified micelles because
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iNGRtꢀmodified micelles can be internalized through NRPꢀ1 mediated process (Figure 6). In
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addition, MDAꢀMBꢀ231 cells treated with iNGRꢀPEGꢀPTX /Cou6 micelles showed slightly
8
higher fluorescence because it is negative on CD13 expression, while it expresses matriptase at a
low level which can cleave cyclic iNGR peptide into linear iNGRt peptide, and realize active
targeting through NRPꢀ1 pathway. The cellular uptake study was also performed on a CD13ꢀ and
NRPꢀ1ꢀnegative cell line HEK293 and there was no significant difference in fluorescence of
HEK293 cells treated with the nonꢀtargeting, iNGRꢀmodified and iNGRtꢀmodified micelles (as
shown in Figure S23).
1
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Figure 5. Cellular uptake of coumarinꢀ6 loaded PEGꢀPTX8 micelles with different modification
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on HUVEC cells (original magnification: 50×). Green: fluorescence signal of coumarinꢀ6.
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Figure 6. Cellular uptake of coumarinꢀ6 loaded PEGꢀPTX8 micelles with different modification
on MDAꢀMBꢀ231 cells (original magnification: 50×). Green: fluorescence signal of coumarinꢀ
6.
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In vitro anticancer efficacy.
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Figure 7. In vitro cytotoxicity of different PTX formulations at various concentrations against
MDAꢀMBꢀ231 tumor cells 48 h after incubation. Data are represented as mean ± SD (n = 4).
3
4
5
Table 2. IC values of different PTX formulations against MDAꢀMBꢀ231 tumor cells (Data are
5
0
presented as mean ± SD, n = 4).
Formulation
Taxol
MeOꢀPEGꢀ
iNGRꢀPEGꢀ
iNGRtꢀPEGꢀ
PTX /PTX
PTX /PTX
PTX /PTX
8
8
8
IC (ng/mL) 76.69 ± 1.27 3774 ± 1.33
1991 ± 1.29
503.6 ± 1.23
5
0
6
7
In vitro cytotoxicity of Taxol, MeOꢀPEG ꢀPTX /PTX, iNGRꢀPEG ꢀPTX /PTX and iNGRtꢀ
5
k
8
5k
8
8
9
0
1
PEG ꢀPTX /PTX micelles against MDAꢀMBꢀ231 cells were investigated by MTT assay. Figure
5k 8
7 displayed that different PTX formulations inhibited cell viability at a doseꢀdependent manner.
1
1
It was shown that the IC50 of iNGRtꢀPEGꢀPTX /PTX micelles (503.6 1.23 ng/mL) was
8
significantly lower than iNGRꢀPEGꢀPTX /PTX micelles (1991 1.29 ng/mL) and MeOꢀPEGꢀ
8
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PTX /PTX micelles (3774 1.33 ng/mL). Cytotoxicity is dependent on the intracellular drug
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1
3
concentration, so the results are consistent with the cellular uptake results. Interestingly, all of
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the micelles have higher IC value than that of Taxol (76.69 1.27 ng/mL) (Table 2), which is
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2
3
possibly because Cremophor EL is also attributed to cytotoxicity
and PTX was not fully
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released from the micelles. Actually, we have investigated the stability of the micelles in cell
culture medium (DMEM+10% FBS+1% PenecillinꢀStreptomycin solution) with MDAꢀMBꢀ231
cells (detailed method was in supporting information), Different micelles were coꢀincubated with
MDAꢀMBꢀ231 cells at an equal PTX concentration (2.5 µg/mL PTX). Results showed that at 1
0
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h, less than 1% of MeOꢀPEGꢀPTX was released while ~7% of MeOꢀPEGꢀPTX /PTX released
8 8
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in cell culture medium with tumor cells, which suggested that the micelles were disintegrated
inside the cells and the physically encapsulated free PTX (5%, w/w) was fully released within 1
h while little conjugated PTX was released (as shown in Figure S24 in supporting information).
The accumulated PTX release of the micelles was not significantly increased until 24 h and the
iNGRꢀmodified micelles showed similar release property, indicating that the carbonate ester
bond remains relatively stable in the medium with MDAꢀMBꢀ231 cells. As the in vitro
cytotoxicity results (Figure 7) displayed that tumor cells were induced to apoptosis by the MeOꢀ
PEGꢀPTX /PTX and iNGRꢀPEGꢀPTX /PTX micelles in a concentrationꢀdependent manner, and
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1
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1
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Figure 12 (see below in the “Antitumor efficacy” section) demonstrated that the micelles have
stronger in vivo antiꢀtumor effect, it could be indirectly concluded that the conjugated PTX could
also induce cell apoptosis in a similar way as free PTX.
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Cell cycle distribution.
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PTX is well known to suppress cell proliferation by inhibiting microtubule disassembly and
arrest cell cycle in the G2/M phase. To investigate the mechanism by which the PEGꢀPTX /PTX
8
micelles inhibit cell proliferation, the population of MDAꢀMBꢀ231 cells in different cell cycle
stages was quantified by FACS analysis of ~10000 cells per sample. Figure 8 concluded the
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results of cell cycle distribution after coꢀincubation with Taxol, MeOꢀPEGꢀPTX /PTX, iNGRꢀ
8
PEGꢀPTX /PTX and iNGRtꢀPEGꢀPTX /PTX micelles at a concentration of 1 µg/mL PTX
8
8
equivalent respectively. Compared to untreated MDAꢀMBꢀ231 cells (control, 13.75% G2/M
phase), Taxol caused a markedly increased accumulation of G2/M phase cells (46.00%)，while
the percentage of cells treated with MeOꢀPEGꢀPTX /PTX micelles, iNGRꢀPEGꢀPTX /PTX
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micelles and iNGRtꢀPEGꢀPTX /PTX micelles in G2/M phase was 32.18%, 30.62% and 39.86%,
8
respectively. iNGRtꢀPEGꢀPTX /PTX micelles induced the strongest cell arrest effect among the
8
groups because cellular uptake efficiency was enhanced by NRPꢀ1 mediating pathway in
accordance with the cellular uptake results. The percentages of cell population in each phase of
the cell cycle are summarized in Table 3.
1
1
1
2
1
Figure 8. Cell cycle analysis of MDAꢀMBꢀ231 cells treated with different PTX formulation at a
1
3
concentration of 1 µg/mL PTX.
14
Table 3. Cell population in each phase of MDAꢀMBꢀ231 cells treated with different PTX
formulations.
1
5
Cell population (%)
G1 Phase
S Phase
G2/M Phase
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Control
Taxol
43.12
15.11
19.20
43.13
38.89
48.63
13.75
46.00
32.18
MeOꢀPEGꢀ
PTX /PTX
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iNGRꢀPEGꢀ
18.34
17.71
51.04
42.43
30.62
39.86
PTX /PTX
8
iNGRtꢀPEGꢀ
PTX /PTX
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In vivo imaging.
In vivo imaging was employed to evaluate the targeting efficiency of BODIPY loaded iNGRꢀ
PEGꢀPTX micelle and MeOꢀPEGꢀPTX micelle in TNBCꢀbearing nude mice. As displayed in
8
8
Figure 9 A, mice treated with iNGRꢀPEGꢀPTX /PTX micelles exhibited stronger nearꢀinfrared（
8
6
NIR）signal at 24 h after intravenously injection through tail vein comparing with MeOꢀPEGꢀ
7
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0
1
PTX /PTX micelles at the tumor site, showing enhanced tumor targeting ability with iNGR
8
peptide modification. 3D image of the tumor site was captured at the same time (Figure 9B).
Moreover, tumors and the major organs were excised for ex vivo imaging to observe the tissue
1
1
distribution (Figure 9C). Tumor of TNBCꢀbearing mice treated with iNGRꢀPEGꢀPTX /PTX
8
micelles also showed stronger signal than that of MeOꢀPEGꢀPTX /PTX micelles. Furthermore,
8
12
NIR fluorescence intensity in normal tissues was much weaker than that in tumors for both
groups, which suggested that besides active targeting, the micelles took good advantage of EPR
effect due to its ultraꢀsmall size and high stability in blood circulation, resulting in reduced
toxicity to normal tissues.
1
1
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4
5
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Figure 9. In vivo biodistribution and targeting effects of different PTX formulations in MDAꢀ
MBꢀ231 bearing mice. (A) In vivo 2D imaging at 24 h after intravenous (iv) injection of MeOꢀ
PEGꢀPTX /PTX micelles (non targeting group) and iNGRꢀPEGꢀPTX /PTX micelles (iNGR
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targeting group) at a dose of 0.2 mg BODIPY/kg in tumor bearing mice (the red dotted circle
was indicated as the location of tumor implantation); (B) In vivo 3D imaging at 24 h after i.v.
injection of iNGRꢀPEGꢀPTX /PTX micelles in tumorꢀbearing mice; (C) Representative ex vivo
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optical images of tumors and organs of tumorꢀbearing mice sacrificed at 24 h.
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Pharmacokinetics and biodistribution.
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The mean plasma concentrationꢀtime profiles of PTX after intravenous administration of
different PTX formulations were plotted in Figure 10, and the corresponding pharmacokinetic
parameters were summarized in Table 4, respectively. As shown in Figure 10, the concentration
of PTX in blood plasma decreased rapidly following the treatment of Taxol. Meanwhile, the
concentration of PTX in blood plasma was maintained for a longer circulation time even after 24
h administration when treated with PTX micelles, due to the stable structure of the micelles. It
was pretty clear that there were significant differences of the pharmacokinetic parameters
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between Taxol and the PEGꢀPTX /PTX micelles, where the mean area under the plasmaꢀ
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concentration curves (AUC ) of MeOꢀPEGꢀPTX micelles and iNGRꢀPEG ꢀPTX /PTX
0
ꢀ∞
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5k
8
11
micelles were 6.52ꢀ and 5.81ꢀfold higher than that of Taxol, respectively, corresponding mean
residence time (MRT) of MeOꢀPEGꢀPTX /PTX micelle and iNGRꢀPEGꢀPTX /PTX micelle was
1
1
1
1
2
3
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5
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5.14ꢀ and 5.17ꢀfold longer than that of Taxol, which suggest a prolonged blood circulation time
of the polymeric micelles than Taxol. However, more studies are needed to fully understand the
drug release and evaluate different fractions of the micelles in the plasma.
16
We next evaluated the in vivo biodistribution of PTX delivered to tumors by systemically
injected MeOꢀPEGꢀPTX /PTX micelle, iNGRꢀPEGꢀPTX /PTX micelle and Taxol at the dose of
1
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1
10 mg PTX/kg. Tissue samples of treated mice were collected by heart perfusion after 2 h and 24
h. Notably, the micelles significantly reduced the drug concentration at other organs, including
heart, liver, spleen, lung and kidney at 2 h (Figure 11A). In particular, compared with Taxol, the
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1
2
level of PTX delivered by MeOꢀPEGꢀPTX /PTX micelles in the heart, liver, spleen, lung and
8
22
kidney was lowered by 20.01ꢀ, 9.44ꢀ, 3.86ꢀ, 10.32ꢀ and 8.28ꢀfold, respectively, and 16.45ꢀ, 3.62ꢀ,
2
3
6.68ꢀ, 6.88ꢀ and 17.41ꢀfold, respectively by that of iNGRꢀPEGꢀPTX /PTX micelles.
8
ACS Paragon Plus Environment
29