Overcoming Multidrug Resistance through GLUT1 Mediated and

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Biological and Medical Applications of Materials and Interfaces

Overcoming Multidrug Resistance through GLUT1 Mediated and Enzyme Triggered Mitochondrial Targeting Conjugate with Redox Sensitive Paclitaxel Release Pengkai Ma, Jianhua Chen, Xinning Bi, Zhihui Li, Xing Gao, Hongpin Li, Hongyu Zhu, Yunfang Huang, Jing Qi, and Yujie Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18437 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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ACS Applied Materials & Interfaces

Overcoming Multidrug Resistance through GLUT1 Mediated and Enzyme Triggered Mitochondrial Targeting Conjugate with Redox Sensitive Paclitaxel Release Pengkai Maa; Jianhua Chena; Xinning Bia; Zhihui Lia; Xing Gaoa; Hongpin Lia; Hongyu Zhua; Yunfang Huanga; Jing Qia; Yujie Zhang* a

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China

*

Address for correspondence: Yujie Zhang

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China, Yangguang South Street, Beijing 102488, China. Tel: 86-010-84738618. Email: [email protected]

Abstract Multidrug resistance (MDR) is thought to be the major obstacle leading to the failure of paclitaxel chemotherapy. To solve this problem, a glucose transporter mediated and matrix metalloproteinase 2

(MMP2)

triggered

mitochondrion

(Glucose-PEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel)

targeting composed

conjugate of

PAMAM

dendrimer and enzymatic detachable glucose-PEG was constructed for mitochondrial delivery of paclitaxel. The conjugate was characterized by 30 nm sphere particle, MMP2 sensitive PEG outer layer detachment from PAMAM and glutathione (GSH) sensitive paclitaxel release. It showed higher cellular uptake both in glucose transporter 1 (GLUT1) overexpressing MCF-7/ADR monolayer cell (2D) and multicellular tumor spheroids (3D). Subcellular location study showed it could specifically accumulate in mitochondria. Moreover, it exhibited higher cytotoxicity against MCF-7/ADR cells, which significantly reverse the MDR of MCF-7/ADR cells. The MDR reverse

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might be caused by reducing ATP content through destroying mitochondrial membrane as well as down-regulating P-gp expression. In vivo imaging and tissue distribution indicated more conjugate accumulated in tumor of the tumor-bearing mice model. Consequently, the conjugate showed better tumor inhibition rate and lower body weight loss, which demonstrated that it possessed high efficiency and low toxicity. This study provides a glucose mediated GLUT targeting, MMP2-responsive PEG detachment triphenylphosponium mediated mitochondria targeting, and GSH sensitive intracellular drug release conjugate that has the potential to be exploited for overcoming MDR of paclitaxel. Keywords: Multidrug resistance; glucose transporter targeting; mitochondria targeting; tumor microenvironment; PAMAM-drug conjugate

1 Introduction Paclitaxel (PTX) and its nanomedicine have been extensively used in clinical for the treatment of breast cancer, lung cancer, ovarian cancer, and so on

1-2

. However, the development of multidrug

resistance (MDR) has significantly limited the therapeutic effect of PTX 3. P-gp, an adenosine triphosphate (ATP)-dependent efflux pump, can pump drugs outward from cancer cells, accounts for the lower accumulation of drug in cytoplasm and final therapy failure 4-5. Mitochondria are the powerhouses of cells by synthesizing ATP via the respiratory chain, targeting delivery of chemotherapeutics to mitochondria to cut off the “energy supply” of P-gp may serves as a promising strategy to diminish MDR. 6-7. To achieve mitochondrial targeted delivery, drug delivery systems (DDSs), such as liposomes, nanoparticles, and PAMAM dendrimer have been designed by the incorporation of mitochondria tropic agents such as mitochondrial targeting signal peptides, lipophilic cations, and protein


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transduction domains

8-10

. These DDSs exhibit preferential mitochondrial accumulation and

increased cytotoxicity in vitro. However, these protocols often bring with low specificity combined with severe non-specific toxicity in system administration

11

. Thus, before considering

mitochondriatropic transportation of drugs, delivering cargos to tumor and tumor cell seems to be an important issue to address 12. The most ubiquitous way to strengthen the delivery efficiency of DDSs is to conjugate targeting ligands, which can specifically identify and bind to the overexpressed receptors on the cancer cell membrane. Alternatively, the transporter-mediated pathways exhibited faster transport rates and better specificity compared with receptor-mediated pathways 13. Facilitative glucose transporter 1 (GLUT1), transferring the D-glucose across cytomembrane, plays a vital role in the glucose transporter protein (GLUT) family 14-15. The proliferation of tumor cells consumes large quantities of glucose 16, leading to the overexpression of GLUT1, which is about 100-fold more plentiful than transferrin receptor. It is proposed that GLUT might be a promising protein target for drug delivery. The oncology research in terms of the design and synthesis of GLUT-targeted anticancer drugs is worthy of attention. 17. As the most typical example, 2-deoxy-2-(18F)-fluoro-D-glucose has been extensively employed as diagnostic tracer for cancer in clinical 18. In addition, the Glufosfamide (glucose conjugated with ifosfamide mustard) shows promising result in end-stage clinical trials for metastatic pancreatic melanoma treatment 19-20. Thus, glucose targeted ligand may be explored to strengthen targeting ability of DDSs for tumor cells. Stimuli-sensitive drug targeting DDSs are capable of delivering drug and releasing drug site-specifically

through responding

to subtle environmental changes between tumor

microenvironments and normal tissues, such as enzyme, pH, ROS


21-23

. Matrix metalloproteinases


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(MMPs), especially MMP2, are over expressed in tumor microenvironment. It has been successfully used to construct size shrinkable or surface changeable DDSs for enhancing tumor penetration or targeting of DDSs. For example, the Au@gelatin nanoparticle (NP) with large size designed by Gao, et al. was first accumulated at tumor and then small sized Au NP with enhanced tumor permeability were released following the gelatin was digested by MMP-2

24

. Hu, et al.

constructed polycaprolactone (PCL) NP covered with PEG of different length, the long PEG chains detach from PCL core following the MMP sensitive peptide linker is cleaved, leading to the exposure of the short PEG chain modified with folic acid, consequently achieving enhanced cellular uptake

25

. Besides that, glutathione (GSH) is a strong biological reducing agent. The

concentration of GSH in normal tissues was more than four times lower than that in tumor tissues. What’s more, the GSH in tumoral intracellular (2-10 mM) is much higher than it in extracellular where GSH concentration is 2–20 µM 26. It has been proved that the disulfide bond can be easily cleaved by intracellular GSH while remaining stable in a predominantly oxidizing extracellular space. Therefore, benefiting from the significant discrepancies in GSH distribution disulfide bonds can be introduced into DDS for GSH sensitive drug release. The rapid drug release produces high intracellular drug concentration, leading to exceed drug efflux thus reversing MDR of cancer cells 27

.

In this study, we rationally designed a mitochondrial targeting polymeric conjugate for overcoming paclitaxel MDR. As shown in Fig. 1, the conjugate is composed of a PAMAM dendrimer polymer core, which is co-modified with mitochondria targeting molecular triphenylphosphine via amido bond and model drug paclitaxel via disulfide bond and further conjugating with long circulating PEG layer via MMP2-sensitive peptide (GPLGIAGQ). Therefore, the conjugates could efficiently


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accumulate in the tumor tissue by EPR effect. Once the system reaching tumor cell via specific interaction between glucose and glucose transporter 1, the PEG layer would detach from PAMAM following the MMP2 sensitive peptide linker was cut off by up-regulated MMP2. Subsequently, the conjugate would target to mitochondria via the triphenylphosphine guidance and paclitaxel would be rapidly released in cytoplasm and mitochondria through reductive reaction. The sufficient high intracellular paclitaxel concentration counteracted the efflux effect of P-gp, on the other side, the paclitaxel directly acted on mitochondria to cut off the energy supply of P-gp, which finally overcome the MDR effect of tumor cells.



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Fig. 1. Schematic illustration of the synthesis route of the Glucose-PEG-peptideTriphenylphosponium-PAMAM-Paclitaxel conjugate and its action mechanism of overcoming MDR. Paclitaxel was conjugated to PAMAM through a 3,3'-dithiodipropionic acid linker to realize reductive sensitive drug release. Then (3-carboxypropyl)-triphenylphosponium bromide was conjugated to PAMAM to realize mitochondria targeting. The terminal of glucosylation PEG (Glucose-PEG-NHS) was first modified with MMP2 sensitive peptide (GPLGIAGQ), and then conjugating

with

PAMAM

to

yield

the

final

conjugate

(Glucose-PEG-Peptide-

Triphenylphosponium-PAMAM-Paclitaxel). The conjugate could passive targeting to tumor tissue


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by EPR effect and active targeting to tumor cell by glucose transporter 1. Subsequently, the PEG layer detached from PAMAM due to the cleavage of peptide linker by extracellular up-regulated MMP2. With the exposure of triphenylphosphine, the conjugate targeted to mitochondria and paclitaxel were rapidly released, which counteracted the pump of P-gp and acted on mitochondria to cut off the power supply of P-gp to overcome the MDR of the cancer cell.

2 Materials and Methods 2.1 Materials PAMAM, paclitaxel (PTX), 3,3'-Dithiodipropionic Acid (DPA), 4-dimethylaminopyridine (DMAP),

1-ethyl-3-(3-dimethylaminopropyl)

(3-Carboxypropyl)

triphenylphosponium

bromide

carbodiimide (TPP),

hydrochloride

N-hydroxysuccinimide

(EDCI), (NHS),

N,N’-Disuccinimidyl carbonate (DSC), and protease inhibitor cocktails were obtained from Sigma (St. Louis, MO. USA). MMP sensitive peptide (GPLGIAGQ) was synthesized by Bank Peptide Inc (Hefei, Anhui Province, China). mPEG-NHS and Glucose-PEG-NHS were purchased from Jenkem Technology Company (Beijing, China). Fluorescent probes were purchased from Beijing FanBo Biochemicals Co., Ltd (Beijing, China). ATP assay kit, JC-1 dyeing working solution, and RIPA lysis buffer were purchased from Beyotime Company (Nanjing, China).

BCA protein assay kit was purchased from Thermo Fisher Scientific Inc. ( Waltham, MA. USA).

2.2 Cell Lines and Tumor Models MCF-7 cell, MCF-7/ADR cell, and L02 cell were gifts from Institute of Pharmacology & Toxicology of Academy of Military Medical Sciences. The establishment of tumor bearing mice model was conducted as described before 28. Briefly, nude mice (20 ± 2 g) were subcutaneously



injected with 200 µL MCF-7/ADR cells (1×107 cells/mL) suspended in PBS (pH 7.0). Experiments were carried out following tumor volume reached 1 cm3. All animal experiments were conducted with the Guide for the Care and Use of Laboratory Animals of Beijing University of Chinese Medicine.

2.3 Synthesis of Glucose-PEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel Conjugate Synthesis of PAMAM-Paclitaxel Conjugate To a solution of PTX (0.24 mmol) and DPA (2.4 mmol) in dichloromethane (DCM) and tetrahydrofuran were added DMAP (0.35 mmol) and EDCI (0.35 mmol). After stirred at room temperature for 24 h, the mixture was concentrated in vacuo and re-dissolved in DCM. The final product was purified by silica column chromatography by eluting with cyclohexane/acetone (3/1) to yield the PTX derivative. To the PTX derivative (18.3 µmol) in DCM were added EDCI (183 µmol) and NHS (200 µmol) and reacted for 24 h to obtain the active ester of PTX. The solvent was removed using the rotary evaporation and the residue was re-dissolved in DMSO and used without further purification. To a solution of PAMAM (3.7 µmol) in pH 8.0 PBS (1 mL) was added PTX active ester (18 µmol) to react for 48 h. The mixture was purified by dialysis against water and then lyophilized to yield the PAMAM-PTX (PTX PAMAM) conjugate.

Synthesis of Triphenylphosponium-PAMAM-Paclitaxel conjugate TPP (0.234 mmol) was added with DSC (0.351 mmol) and triethylamine in DCM to react for 24 h to yield TPP active ester 29. Then, the TPP active ester was added to PP conjugate (3.7 µmol) in DMSO to react for 48 h. The DMSO was removed by dialysis against water and then lyophilized to yield the Triphenylphosponium-PAMAM- Paclitaxel (TPP/PTX PAMAM) conjugate.


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Synthesis

of

Glucose-PEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel

conjugate

Glucose-PEG-NHS (100 µmol) in pH 8.0 PBS was added MMP sensitive peptide (GPLGIAGQ, 150 µmol) and put on vortex mixer for 2 h to synthesize the Glucose-PEG-GPLGIAGQ (GPp) conjugate. Then, free peptide was removed by dialysis and the residue was reacted with TPP conjugate (3.7 µmol) in pH 8.0 PBS (1 mL) for 48 h. The mixture was dialyzed against water for 24 h

and

then

lyophilized

to

Glucose-PEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel conjugate

29

.

The

mPEG

was

also

yield

the

(GPp/TPP/PTX

PAMAM)

used

synthesize

to

mPEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel (mp/TPP/PTX PAMAM) conjugate with the same method. Fluorescent dyes (FITC and Cy7) labeled conjugates were also synthesized with the method described before to evaluate the cellular uptake and bio-distribution in whole body

29

. The PTX

derivative was determined using high resolution mass spectrum (HRMS) and conjugates were analyzed using 1HNMR.

2.4 Characterization of Conjugates 2.4.1 Size, Zeta Potential and Morphology A dynamic light scattering (DLS) particle size analyzer (Nicomp 380 ZLS; PSS, Port Richey, FL, USA) was employed to analyze the nanoparticle size, zeta potential and polydisperse index (PDI) of various conjugates (10 mg/mL) in ultrapure water. The GPp/TPP/PTX PAMAM conjugate with or without peptide linker was co-cultivated by MMP2 (1 mg/ml) for 24 h to evaluate the matrix metalloproteinase 2 (MMP2) sensitive particle size change. The transmission electron microscopy (TEM) (JEM 1400 JOEL, Tokyo, Japan) was employed to observe the morphology of conjugates



Page 10 of 33

following staining with a 2% sodium phosphotungstate solution.

2.4.2 MMP Sensitivity An enzymatic digestion method was employed to evaluate the MMP2 sensitivity of MMP2-sensitive peptide (GPLGIAGQ) and its conjugates as described before

30-31

. Briefly, 1

mg/mL peptide, Glucose-PEG-peptide, and Glucose-PEG-peptide-PAMAM were incubated with active human MMP2 with different concentrations (0 ng/µL , 1 ng/µL and 10 ng/µL) at 37 °C for 24 h with gentle shaker. The reaction mixture was monitored by RP-HPLC with a Purospher STAR RP-18e column at a flow rate of 1 mL/min (water/acetonitrile = 10/1, V/V) and detected at 280 nm.

2.4.3 GSH Sensitive Drug Release The GSH reduction sensitive release profile of PTX from the GPp/TPP/PTX PAMAM conjugate was studied using dialysis method. The conjugate was dissolved in water (containing 1 mg PTX) and transferred into dialysis bags (MWCO 3500 Da) and dialyzed against 30 ml pH 7.4 PBS containing 0.5% Tween-80 and different concentration of GSH (0 mM, 10μM and 10 mM). The drug release of GTPP conjugate without disulfide bond was also explored as control. The study was performed at 37 °C in a water bather with gently shaken at 100 rpm. At predefined time, 1 mL external medium was sampled and the content of PTX was determined using HPLC at 230 nm with the mobile phase consisting of acetonitrile/water (50:50, v/v).

2.5 GLUT1 Targeting Evaluation The in vitro GLUT1 targeting capacity of the conjugates was assessed by carrying out cellular uptake of conjugates by GLUT1 over-expressing MCF-7/ADR cells and low-expressing L02 cells. Briefly, under the environment with 5% CO2, cells were cultivated for 24 h at 37°C in 96-well plates with a concentration of 5×103 cells/well. After reaching 70-80% confluence, cells were


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co-incubated with FITC labeled conjugates for different time (1 h, 4 h, 8 h) at a concentration of 1

μ M. To further confirm the GLUT1 targeting specificity of the GPp/TPP/PTX PAMAM conjugate, the GLUT1 transporter of MCF-7/ADR cells was blocked by pre-incubating with 2.5 mM D-glucose (D-GLU) for 4 h. For qualitative analysis cells were

fixed with

paraformaldehyde at a concentration of 4% and observed using a fluorescence microscopy (TE-200, Nikon, Tokyo, Japan); for quantitative analysis the cells were trypsin digestion, washed three time with PBS and analyzed by flow cytometry (BD FACSAria III, BD Biosciences, San Jose, CA, USA). Besides that, the cellular uptake was also conducted on a multicellular tumor spheroids (MCTS) model. MCTS model was constructed using the method previously depicted with little modification 32. Briefly, MCF-7/ADR cells were seeded in 2% agarose coated 96-well plates with a density of 2×103 cells/well and incubated for 5 days to form compact sphere. Then, culture medium was replaced with 1 µM TPP/PTX PAMAM, mp/TPP/PTX PAMAM, and GPp/TPP/PTX PAMAM conjugates and co-incubated for 24 h. Thereafter, a confocal laser scanning microscope (CLSM) (Olympus FV1000, Japan) was employed to observe the MCTS at a depth of 100 µm (about the middle position of the MCTS).

2.6 Mitochondrial Targeting Evaluation Subcellular localization of conjugates was observed by CLSM to assess the mitochondrial targeting capacity of conjugates. The MCF-7/ADR cells were seeded on a cover glass which embedded in the bottom of 24-well plates at a concentration of 1×105 cells/well. The cells were co-incubated with the conjugates for 1 h at a concentration of 1µM. Thereafter, the mitochondrion was stained by 50 nM MitoTracker® Deep Red FM for 30 min, and the nucleus was stained by 10



nM Hoechst 33342 for 30 min.

2.7 Cell viability MTT assay method was used to evaluate the cytotoxicity of various blank conjugates and drug loaded conjugates on MCF-7 and MCF-7/ADR cells. The cells were seeded in 96 well plates at a concentration of 5×103 cells/well. After reaching a confluence of 70-80%, drug solution was added at different concentrations and co-incubated with cells for 48 h. Afterwards, 20 µL MTT solution was added at a concentration of 5 mg/mL and co-incubated with cells for 4 h, and 150 µL DMSO was added to dissolve the forming formazan crystal. The absorbance at 490 nm of each well was determined and the following formula was used to calculate the cell viability Cell Viability = Adrug/Acontrol × 100%.

2.8 Mechanism of Reversal MDR To determine intracellular ATP content, MCF-7/ADR cells were placed into 12-well plates at a concentration of 1×105 cell/well and cultured for 24 h, and then different conjugates containing 5 µM PTX were added in and co-incubated with the cells were for 8 h at 37 °C. The cells treatment with drug free culture medium was employed as negative control. An ATP assay kit was used to determine the intracellular ATP content. To determine the mitochondrial membrane potential

(MMP), MCF-7/ADR cells were co-incubated with conjugates for 24 h, and then discard the medium, trypsin digestion, washed three times with PBS, collected and then treated with JC-1 dyeing solution

33

. The fluorescence intensity was determined using a ﬂow cytometry. Western

blot was used for P-gp content analysis. Briefly, following cells co-incubated with various conjugates for 24 h, the total protein of cells was extracted by RIPA lysis buffer containing protease inhibitor cocktails. A BCA protein assay kit was used to determine the protein


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concentration. The protein concentration was adjusted to 10 mg/ml and 0.2 ml was loaded for western blot analysis with β-actin as a loading control. The protein band was captured using a Bio Imaging System (Clinx, China) and analyzed using Image J software.

2.9 In Vivo Targeting Evaluation Conjugates were intravenously administered via the tail vein at a Cy7 dose of 1 mg/kg. Following anesthetization with diethyl ether, an in vivo imaging system (Carestream Fx Pro, NY, USA) was applied for determining the fluorescence from conjugates at predefined intervals of 1, 4, 8 and 12 h post-injection. In order to quantify the fluorescence intensity of conjugates in tissues, mice were sacrificed using carbon dioxide and tissues (including heart, liver, spleen, lung, kidney, and tumor) were harvested and collected to observe. The fluorescence intensity was analyzed by the carestream software.

2.10 Tumor Growth Inhibition The in vivo anti-tumor effect of conjugates was performed on tumor-bearing nude mice. The mice were randomly divided into six groups following tumor volume reached approximately 100 mm3and intravenously injected with physiological saline, PTX solution, and different conjugates at a PTX dose of 5 mg/kg. Mice were administered drug every 2 days for 14 days. The body weight of mice was recorded every other day to evaluate the systematic toxicity of different formulations. After mice were sacrificed, tumors were collected and weighted. The inhibition ratio was calculated according to the following formula: tumor inhibition ratio =（1-T/C）×100%，T represented the average weight of treated groups, and C represented the average weight of control group.



2.11 Statistical Analysis Data were showed as the average ± SD.

Statistical significance between groups was

analyzed using SPSS statistical software and when P ＜ 0.05, it was regarded as statistically significant.

3 Results and Discussion 3.1 Synthesis of Glucose-PEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel Conjugate The determined molecular mass for paclitaxel derivative and MMP2 sensitive peptide (GPLGIAGQ) were 711.35 and 1045.31 respectively, as shown in Fig 2A, which were consistent with their theoretical molecular mass. The structure of different conjugates were confirmed by 1

HNMR, as shown in Fig. 2B-2F, the peaks at δ (ppm) ≈2.2–3.4 could be assigned to the

methylene protons of the branching units of PAMAM

34

. The peaks at δ (ppm) ≈ 7-8 were

characteristic peaks of phenyl groups belonging to PTX and TPP, which demonstrated they were successfully linked to PAMAM. The appearance of signals at δ (ppm) ≈0.5–2.0 and 3.6 belong to aliphatic protons of peptides and the methylene protons of PEG, respectively, which indicated the glucose-PEG-peptide was successfully synthesized. All the above peaks could be found in the 1

HNMR of GPp/TPP/PTX PAMAM. The number of PTX and TPP conjugated to PAMAM was

determined to be 3 and 10 respectively, by integrating the aromatic peaks located at δ 7-8. The conjugated PEG calculated to be 20 by integrating the methylene peaks located at δ 3.6 35.


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Fig. 2. HRMS of (A) MMP2 sensitive peptide GPLGIAGQ (positive mode) and PTX derivative (negative mode); 1HNMR of (B) PAMAM, (C) PTX PAMAM, (D) TPP/PTX PAMAM, (E) GPp and (F) GPp/TPP/PTX PAMAM.

3.2 Characterization of Conjugates 3.2.1 Size, Zeta Potential and Morphology The particle size and surface charge have significant implications on not only the cellular uptake but also the bio-distribution of DDS 36. As shown in Fig.3, the particle sizes for PTX PAMAM, TPP/PTX



PAMAM, mp/TPP/PTX PAMAM, and GPp/TPP/PTX PAMAM conjugates were 30 nm, 33.6 nm, 40.3 nm, and 42.5 nm, respectively. The particle size increased with conjugation with more molecules. All particle sizes were below 50 nm, which were large enough for passive targeting that based on the EPR effect. As for zeta potential were 6.2 mV, 8.9 mV, 2.5 mV, and 2.9 mV respectively. The PEG modification effectively neutralized the positive charge on the surface of PAMAM, which is essential for escaping nonspecific interaction with negative charge of blood cell membrane in whole body circulation

37

. Meanwhile, the particle size decreased and zeta potential increased following

co-incubated with MMP2, which was due to the release of PEG-peptide. It could be inferred that the conjugate modification with GPLGIAGQ peptide was sensitive to MMP2. The morphology observed by TEM revealed that all conjugates were mono-dispersed with spherical shape. Whereas, the particle size determined by TEM was smaller than the DLS result. It might be caused by the hydrated corona

of conjugates when determined by DLS in water. Obvious particle size decrease of peptide linked conjugate was also observed by TEM following treatment with MMP2, while no change for non-peptide linked conjugate, which confirmed that the peptide linker was MMP2 sensitive. The characteristics of different conjugates were summarized in Table 1.


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Fig. 3. Particle size and morphology of PTX PAMAM (A, D), TPP/PTX PAMAM (B, E), mp/ TPP/PTX PAMAM (C, F), and GPp/TPP/PTX PAMAM (G, J) conjugates analyzed by DLS and TEM, and the particle size and morphology change of GPp/TPP/PTX PAMAM conjugate with (H, K) or without (I, L) MMP2 sensitive peptide after treatment with MMP2. Table 1. Physicochemical characteristics of different conjugates Number

PTX

TPP

mPEG

Size (nm)

GLU-PEG

Zeta Potential (mV)

PDI

DLS

TEM

30.0±12.5

6.26±1.38

6.2±1.3

0.15±0.03

33.6±14.2

6.49±1.27

8.9±1.7

0.11±0.02

40.3±20.5

6.82±1.67

2.5±0.6

0.16±0.02

PTX PAMAM

3

TPP/PTX PAMAM

3

10

mp/TPP/PTX PAMAM

3

10

GPp/TPP/PTX PAMAM

3

10

20

42.5±18.4

8.87±2.58

2.9±1.1

0.15±0.04

GPp/TPP/PTX PAMAM (with MMP2)

3

10

20

37.2±17.6

6.04±1.19

7.6±1.5

0.16±0.03

GPp/TPP/PTX PAMAM (without MMP2)

3

10

20

48.5±25

13.23±3.74

3.4±1.3

0.18±0.06

20

3.2.2 MMP Sensitivity and Reductive Drug Release The MMP sensitivity of the peptide, PEG-peptide, and PEG-peptide-PAMAM conjugate was further evaluated by HPLC following enzymatic digestion. As shown in Fig. 4A, the peak of the peptide reduced after incubation with MMP2 at low or high concentration, and the peak reduced more when



treatment with higher concentration of MMP2, which indicated that the peptide GPLGIAGQ was MMP2 sensitive and could be cleaved into two fragments GPLG and IAGQ. Besides that, the glucose-PEG-peptide, and glucose-PEG-peptide-PAMAM conjugate showed similar MMP sensitivity compared with peptide alone, which demonstrated that MMP2 could contact with and cleave the peptide even after conjugation with PEG or PAMAM. Furthermore, the reductive sensitive drug release of the GPp/TPP/PTX PAMAM conjugate was investigated under different concentration of GSH, which was used to mimic different physiological environment in vivo. As shown in Fig. 4D, 26% or 52% drug was released from the conjugate within 12 h under normal physiological condition (0 or 10 µM GSH), which was far lower than 79% under tumor cellular micro-environment (10 mM). Moreover, the conjugate without disulfide bond also showed significantly slow drug release with less than 36% in 48 h. These results indicated that the conjugate was stable and has no drug burst release when in whole body circulation. Whereas, the conjugate exhibited significantly accelerated drug release when entering into the tumor cell and exposing to high concentration of GSH.


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Fig. 4. Enzymatic cleavage MMP2 sensitive (A) GPLGIAGQ peptide, (B) Glucose-PEG-peptide and (C) Glucose-PEG-peptide-PAMAM conjugate, the samples were treated with 5 ng/µL MMP2 for 24 h followed by HPLC determination; (D) In Vitro drug release of GTPP conjugate under different reductive environment, the GPp/TPP/PTX PAMAM conjugate without disulfide bond linker was used as control.

3.3 In Vitro GLUT1 Targeting Evaluation Cellular uptake was qualitatively and quantitatively analyzed on both monolayer cell and MCTS models to evaluate the targeting specificity and internalization efficiency. As shown in Fig. 5A and B, the fluorescence intensity of all conjugates increased along with the lapse of time, indicating the cellular uptake was time dependent. The TPP/PTX PAMAM conjugate showed higher fluorescence intensity compared with mp/TPP/PTX PAMAM conjugate. It was mainly due to the hydrophily and charge screen effect of PEG corona which hampered the cellular uptake of mp/TPP/PTX PAMAM conjugate. The fluorescence intensity of GPp/TPP/PTX PAMAM conjugate was higher than mp/TPP/PTX PAMAM for MCF-7/ADR cells and the superiority could be counteracted by pretreatment with free glucose. Additionally, the cellular uptake of GPp/TPP/PTX PAMAM conjugate for MCF-7/ADR cells (GLUT1 over-expressing) was more efficient than L02 cells (GLUT1 low-expressing), demonstrating the specific interaction between glucose and GLUT1. The in vitro targeting evaluation was further conducted on a MCTS model, which could better mimic the tumor microenvironment

38-39

. As shown in Fig 5C and 5D, the fluorescence intensity for

mp/TPP/PTX PAMAM was similar with TPP/PTX PAMAM conjugate, indicating the PEG could detach from PAMAM when entering tumor microenvironment. The GPp/TPP/PTX PAMAM conjugate showed highest fluorescence intensity among all conjugates and could be blocked by free



glucose, which was in accordance with the monolayer cell model, indicating a significant synergistic action of GLUT1 targeting and PEG detachment. The intracellular localization was conducted to assess the mitochondrial targeting capacity of different conjugates. The green fluorescence derived from TPP/PTX PAMAM and GPp/TPP/PTX PAMAM conjugates overlapped with the red fluorescence of mitochondria, indicating they accumulated in the mitochondria after entering cell (Fig. 5E). While, the GPp/TPP/PTX PAMAM conjugate exhibited higher efficiency, demonstrating the GLUT1 targeting contributed to mitochondria targeting.


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Fig 5. (A,B) Qualitative and quantitative cellular uptake of conjugates for MCF-7/ADR cells and L02 cells at 1 h, 4 h, and 8 h; (C) Confocal laser scanning microscope images of MCF-7/ADR MCTS after treating with

1 µM conjugates for 1, 4, and 8 h, respectively; (D) Quantitative MCTS uptake at

different times; (E) Subcellular localization of conjugates in MCF-7/ADR cells visualized by confocal fluorescence microscopy. The green fluorescence is derived from FITC, blue fluorescence is from nuclei and red fluorescence is from mitochondria, respectively. The white arrows represent the conjugates were co-localized with the mitochondria. *p < 0.05.

3.4 Cell Viability MCF-7 cells overexpress GLUT1 transporter, and MCF-7/ADR cells overexpress both GLUT1 and


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P-gp transporters. The MTT assay was performed on both cells.. As shown in Fig. 6A and 6B, the non-PEGylation blank conjugates showed significant cytotoxicity to both cells, which might derived from the nonspecific interaction between positive charge of conjugates and negative charge of cell membrane. Whereas, the PEGylation blank conjugates showed significant lower cytotoxicity, which revealed that the PEGylation could shield positive charge to overcome its nonspecific cytotoxicity

40

.

The cytotoxicity of the drug loaded conjugates, as shown in Fig. 6C and 6D, the TPP/PTX PAMAM conjugate showed higher cytotoxicity than mp/TPP/PTX PAMAM, which might be due to the synergistic effect of positive charge of the conjugate and mitochondria targeting ability derived from TPP/PTX PAMAM. As shown in Tab.2, the IC50 of TPP/PTX PAMAM, mp/TPP/PTX PAMAM, and GPp/TPP/PTX PAMAM conjugates for MCF-7 cells were 0.15, 0.92, and 0.093 nM respectively, and 0.84, 8.56, 0.49 nM for MCF-7/MDR cells. The GPp/TPP/PTX PAMAM conjugate showed strongest cytotoxicity and MDR reversal efficacy, which demonstrated that the GPp/TPP/PTX PAMAM conjugate could be internalized in cell via GLUT1 mediated endocytosis and rapidly release drug to cause cytotoxicity as well as bypass the P-gp pump via mitochondria targeting.



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Fig. 6. Dose-dependent cytotoxicity of blank conjugates (A, B) and drug loaded conjugates (C, D) against PTX sensitive MCF-7 cells and PTX resistant MCF-7/ADR cells at 48 h. The experiment was repeated at three times and each experiment was carried out in triplicate. Data are expressed as average ± SD. Table 2. The conjugates reverse PTX resistance in MCF-7/ADR cells after 48 h treatment Formulations

MCF-7

MCF-7/ADR

IC50 Value (nM)

FR

IC50 Value (nM)

FR

Reversal index

Free PTX

1.54

1.00

87.78

57.00

TPP/PTX PAMAM

0.15

0.10

1.30

0.84

67.86

mp/TPP/PTX PAMAM

0.92

0.60

13.18

8.56

6.66

GPp/ TPP/PTX PAMAM

0.093

0.06

0.75

0.49

116.33

FR: fold-resistance was defined as ratio of IC50 value for PTX conjugates to IC50 value for free PTX.

3.5 Mechanism of MDR Reversal As well known, P-gp, an efflux pump transporter, takes charge of regulating the entering of drugs into the cell

41

. Thus, the expression level and activity of P-gp will significantly influence the


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MDR effect. After confirming the GPp/TPP/PTX PAMAM conjugate could target to mitochondria and reverse MDR effect, the potential MDR reversal mechanism was investigated. First, the inﬂuence on the ATP production in MCF-7/ADR cells was evaluated, as shown in Fig. 7A, the free PTX slightly decreased intracellular ATP level. While, cells treated with conjugates showed remarkably decreased ATP level. Moreover, the GPp/TPP/PTX PAMAM conjugate showed highest inhibition efficacy on ATP production and the effect could be impaired by free glucose. Due to the ATP was produced by mitochondria, so the influence on mitochondria was further evaluated. As shown in Fig. 7B, the mitochondria membrane potential (MMP) changing trend was similar with the ATP content, the GPp/TPP/PTX PAMAM conjugate exerted strongest effect and the effect could be counteracted by free glucose. These data indicated that the GPp/TPP/PTX PAMAM conjugate could disturb the mitochondrial function through mitochondrial depolarization and reducing intracellular ATP production. Besides, the western blot analysis was conducted to evaluate the expression of P-gp after treating with different conjugates. As shown in Fig. 7C and D, compared with MCF-7 cells, the expression level of P-gp in MCF-7/ADR cells was higher and it significantly decreased after treatment with conjugates. Additionally, the GPp/TPP/PTX PAMAM conjugate showed higher inhibition efficacy than the TPP/PTX PAMAM and mp/TPP/PTX PAMAM conjugates. The TPP/PTX PAMAM conjugate and the mp/TPP/PTX PAMAM conjugate showed comparative inhibition efficacy. It could be inferred that the GPp/TPP/PTX PAMAM conjugate could reverse the MDR effect of MCF-7/ADR cells, and the mechanism might lie on the reduction of P-gp expression companied with the suppression of the P-gp activity through ATP deprivation and MMP destruction, while the latter had a major role.



Fig.7. ATP content (A), mitochondrial membrane potential (B), and western blot analysis for P-gp expression (C) and corresponding quantitative analysis (D) following treating with various conjugates in MCF-7/ADR cells. *P

Overcoming Multidrug Resistance through GLUT1 Mediated and

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