Biomaterials 59 (2015) 88e101

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Synthesis of a novel, sequentially active-targeted drug delivery nanoplatform for breast cancer therapy Arpan Satsangi a, b, *, Sudipa S. Roy c, Rajiv K. Satsangi d, Anthony W. Tolcher e, Ratna K. Vadlamudi c, Beth Goins f, Joo L. Ong b a

Joint Graduate Program in Biomedical Engineering, The University of Texas at San Antonio and the University of Texas Health Science Center at San Antonio, San Antonio, TX 78249, United States Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX 78249, United States c Department of Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States d RANN Research Corporation, San Antonio, TX 78250, United States e START e South Texas Accelerated Research Therapeutics, LLC, San Antonio, TX 78229, United States f Department of Radiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, United States b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 December 2014 Received in revised form 15 March 2015 Accepted 23 March 2015 Available online

Breast cancer is the leading cause of cancer deaths among women. Paclitaxel (PTX), an important breast cancer medicine, exhibits reduced bioavailability and therapeutic index due to high hydrophobicity and indiscriminate cytotoxicity. PTX encapsulation in one-level active targeting overcomes such barriers, but enhances toxicity to normal tissues with cancer-similar expression profiles. This research attempted to overcome this challenge by increasing selectivity of cancer cell targeting while maintaining an ability to overcome traditional pharmacological barriers. Thus, a multi-core, multi-targeting construct for tumor specific delivery of PTX was fabricated with (i) an inner-core prodrug targeting the cancer-overexpressed cathepsin B through a cathepsin B-cleavable tetrapeptide that conjugates PTX to a poly(amidoamine) dendrimer, and (ii) the encapsulation of this prodrug (PGD) in an outer core of a RES-evading, folate receptor (FR)-targeting liposome. Compared to traditional FR-targeting PTX liposomes, this sequentially active-targeted dendrosome demonstrated better prodrug retention, an increased cytotoxicity to cancer cells (latter being true when FR and cathepsin B activities were both at moderate-to-high levels) and higher tumor reduction. This research may eventually evolve a product platform with reduced systemic toxicity inherent with traditional chemotherapy and localized toxicity inherent to single-target nanoplatforms, thereby allowing for better tolerance of higher therapeutic load in advanced disease states. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Sequential active-targeting Dendrimer Liposome Breast cancer Drug delivery Nanoparticle Chemotherapy Nanoencapsulation

1. Introduction Globally, breast cancer is the most frequently diagnosed cancer in women and the leading cause of cancer death among women [1]. In 2012, it was estimated that 1.7 million new cases were reported and about 522,000 breast cancer deaths occurred in women. Chemotherapy plays a central role in cancer treatment, and the scientific advances in chemotherapy during the last few decades have led to significant improvements in patient survival rates. However, common anti-cancer agents demonstrate well-known limitations, such as systemic cytotoxicity, poor solubility in body * Corresponding author. 7795, Mainland Drive, Suite 103, San Antonio, TX 78250, United States. Tel.: þ1 210 887 2006. E-mail address: [email protected] (A. Satsangi). http://dx.doi.org/10.1016/j.biomaterials.2015.03.039 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

fluids, and slow tumor uptake [2]. Nanotechnological advancements are being applied to overcome these problems. This report is one such effort towards targeted delivery and improved efficacy of an anti-cancer agent commonly used for the treatment of breast cancer. The broadly-cytotoxic class of drugs ‘taxanes’, which includes paclitaxel (PTX) and docetaxel, is the most effective single-agent drug class used in the chemotherapy of breast cancer and the first-line therapy in the metastatic form of the disease [3,4]. PTX is used preferentially [5], with a 56% response rate against metastatic breast cancer [6,7]. Therefore, PTX was used as a model anti-cancer drug in this study. Anti-cancer treatment involving PTX faces several limitations, including reduced bioavailability due to high hydrophobicity. Cremophor EL formulations improve solubility, but introduce severe

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side effects [6,8e10]. Further, the antitumor efficacy of many clinical anti-cancer drugs, including PTX, is limited by their nonspecific, indiscriminate distribution among all tissues and the non-specific cytotoxicity to all proliferating cells (cancerous as well as normal), resulting in significant toxicity, a low therapeutic index, and a narrow therapeutic window. In particular, PTX binds to microtubules within a dividing cell during mitosis, causing kinetic stabilization through inhibition of microtubule depolymerization. The result is mitotic arrest and blockage of cell cycle progression [11e13]. This mechanism is non-specific and can produce serious unwanted consequences. Nanotechnological advances provide for an alternative solution to enhance the bioavailability of potent anti-cancer drugs at the site of their action, while reducing drug toxicity. Shortcomings in drug delivery have given impetus to develop a targeting approach towards (i) the specific physicochemical characteristics of tumor cells and tissue, such as leaky tumor vasculature (passive targeting), and/or (ii) the biomolecules that are differentially expressed in the tumor cell or on their surface relative to the normal cells (active targeting). The targeted drug-carrying platforms that ferry the drug to the tumor increase the bioavailability of the drug at the site of action while limiting exposure to non-target tissues and organs [14,15]. While targeting of a specific molecular cue on cancer cells can prevent systemic toxicity by reducing the number of potential hits at non-cancer cells, normal tissue with similar molecular expression profiles are still exposed to the cytotoxic risk. Thus, the toxicity to non-cancerous yet similarly-expressive normal cells may remain unchanged in a single-target approach and may result in serious toxicity to one or more other vital functions [16]. Therefore, a need exists to develop a multi-targeted drug delivery platform that may be suitable to overcome physiological barriers and enhance specificity to breast cancer treatment and its efficacy [17]. Here, we describe the design of a multi-core drug delivery platform that offers the possibility of multi-focused targeting of breast cancer tissue and cells. The inner core of this construct consists of a PTX delivery device that releases PTX following the action of a proteolytic enzyme upregulated in breast cancer cells. We have previously described the synthesis of such a prodrug [18]. Intended to release PTX in the presence of cathepsin B overexpression (as is the case in many cancer cells, but not normal tissue), it is designed by the conjugation of PTX to the terminal amino groups of the polyamidoamine (PAMAM) dendrimers through the cathepsin B-cleavable tetrapeptide, GlycinePhenylalanine-Leucine-Glycine (GFLG). This prodrug, referred to as PGD (Fig. 1), demonstrated successful cytotoxic and antitumor efficacy [18]; yet by design, the conjugate possesses a single-level targeting capability only, limiting its potential due to the aforementioned cytotoxic effects seen in such platforms. Encapsulation of the PGD in a liposomal outer core allows for the development of a sequentially-targeted multicore architecture (Fig. 2). Liposomes are phospholipid (PL) bilayered vesicles with a membrane mimicking architecture, including a hollow structure to allow for the enclosure of drug warheads. Several liposomal compositions have evolved to include evasion of the reticuloendothelial system (RES) [19,20] and active targeting to cell surface receptors [21,22]. In this study, we have designed a formulation that includes polyethylene glycol (PEG)-based RES evasion and folate receptor (FR)-targeting. To introduce active targeting in the liposomes, the distal end of PEG was conjugated to folate, one of the most widely studied small molecules as a cancer cell targeting moiety. Folate is essential for rapid cell division and growth, and has a high binding affinity for FR (Kd ¼ 109 M). In cancers, since FRs are overexpressed on tumor cells, the folate moiety enables the targeted delivery of therapeutic agents to tumors [23].

89

O O NH

O

O

O

OH

O O

O

HO O O O

O O

O

HN O

Paclitaxel NH O

HN O NH N H

O GFLG

PAMAM Dendrimer Fig. 1. Structure of paclitaxel-GFLG-PAMAM dendrimer (PGD) conjugate (image components not to scale).

While paclitaxel-based liposomal formulations that target FRs exist [24], they display strong limitations: (i) active targeting at a single-level only and (ii) component leakage before reaching target site [25]. To overcome disadvantages of both the dendrimer and liposome platforms yet exploit their advantages, the dendrosome construct was designed to sequentially exploit its potential for (i) long-term circulation, (ii) protection of the inner core prodrug from the circulatory biological environment until it reaches the target site, (iii) passive targeting to take advantage of the enhanced permeation and retention (EPR) effect in tumor tissue caused by chaotic and leaky vasculature along with dysfunctional lymphatic drainage, (iv) active targeting of upregulated FRs on rapidly dividing cancer cells for internalization of prodrug in cancer cells, and (v) further active targeting of cathepsin B, a highly upregulated enzyme in many breast cancer cells to liberate the active agent PTX from its dendrimer-based prodrug. It was expected that the sequential active targeting is achieved such that inner core targeting is not activated until outer core targeting requirements are met. Thus, the overall goal was to improve the therapeutic efficiency of the active agent and to achieve increased targeting specificity using two molecular differences between cancer and normal cells instead of just one. Therefore, this report describes the design and synthesis of the above dendrosome architecture (hereafter referred to as FRtargeting PGD dendrosomes), its physicochemical characterization, and subsequent evaluation through in vitro and in vivo experiments. The device was evaluated with respect to traditional FRtargeting PTX liposomes for improved efficacy in treating cancer cells and reduced toxicity against normal cells. Confirmation of increased treatment efficacy by FR-targeting PGD dendrosomes was evaluated by investigating tumor growth reduction in xenograft mice models of breast cancer.

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Fig. 2. Dendrosome-based sequential active-targeting strategy. Dendrosome targets cancer-specific cell surface receptor, is captured by cell, and allows for intracellular release of targeting dendrimer. Cancer-specific enzyme releases drug from dendrimer-drug conjugate by cleaving substrate-peptide linker.

2. Materials and methods 2.1. Chemical syntheses 2.1.1. Synthesis of paclitaxel-GFLG-dendrimer conjugate (PGD) PGD synthesis was performed as reported elsewhere earlier [18].

2.1.2. Folate bound poly(ethylene glycol)distearoylphosphatidylethanolamine (FA-PEG-DSPE) The synthesis of FA-PEG-DSPE was performed following the method of Gabizon et al. [26]. Briefly, folic acid (FA; 100 mg, 0.244 mmol; Sigma Aldrich) was reacted with Amino-PEG2000DSPE (400 mg, 0.14 mmol; Sigma Aldrich) in anhydrous DMSO (4 mL; Sigma Aldrich) in basic medium. The coupling was affected

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by addition of dicyclohexylcarbodiimide (DCC; 130 mg, 0.63 mmol; Sigma Aldrich). The mixture was stirred magnetically under nitrogen atmosphere. A 5 mL fraction of reaction was removed every 24 h and freeze dried, and the residue was monitored by TLC for the consumption of amino-PEG-DSPE. When amino-PEG-DSPE was completely consumed, the remaining reaction mixture was lyophilized and the residue was partitioned between water and chloroform. The organic extract was dried under a stream of nitrogen and the residue was dissolved in a mixture of DMSO/H2O (80:20) and dialyzed against the same solution. The pure product (385 mg; 87% recovery) was obtained as a yellow solid by lyophilizing the dialysate. TLC: [silica gel-GF/(CHCl3/CH3OH/H2O; 75:36:6)] Rf e 0.56; 1 H NMR [DMSO-d6: referenced to d 0.00 ppm for TMS as internal standard]: d 0.87 (t, 6H, terminal CH3 of stearoyl groups), d 1.24 (s, 56H, C4 through C17 CH2 of stearoyl groups), d 1.55 (m, 4H, CH2CH2CO of stearoyl groups), d 2.20e2.39 (m, 8H, CH2CH2CO of stearoyl groups and CH2 of Glyceryl moiety), d 3.3 (m, 4H, CH2CH2N), d 3.53 (s, xxxH, PEG protons), d 4.00 (t, CH2OCONH, 2H), d 4.1e4.3 (2xdd, trans-PO3CH2CH, cis-PO3CH2CH, 2H þ 2H), d 4.35e4.37 (m, 1H, a-CH-Glyceryl), d 4.54 (d, 2H, 9-CH2N), d 5.22 (m, PO3CH2CH, 1H), d 6.65 (d, 2H, 30 ,50 -Aromatic protons), d 7.68 (d, 2H, 20 ,60 - Aromatic protons), d 8.77 (s, 1H, C7-H). 2.2. Preparation of liposomal encapsulations Liposomes were prepared under sterile conditions using the previously described thin-film hydration method [27]. Lipid components of the FR-targeted liposomes were egg phosphatidylcholine (ePC; from Sigma Chemical Co. St. Louis, MO), cholesterol (Sigma), PEG-DSPE (from Avanti Polar Lipids Inc., Albaster AL), and FA-PEG-DSPE at molar ratios of 8:2:0.24:0.06. PTX (ChemieTek, Indianapolis, IN) or PTX-equivalent PGD was utilized at a paclitaxelto-lipid molar ratio of 1:35. Lipid and drug ingredients were taken in a mixture of organic solvents in a 100 mL capacity round bottom flask. When solvents were completely removed, the lipid film was hydrated with a sucrose-HEPES-saline buffer (300 mM sucrose, 150 mM NaCl, 10 mM HEPES, adjusted to pH 6.5), followed by sonication to ensure complete film detachment. The lipid suspension was extruded through a series of polycarbonate filters of sequentially decreasing pore sizes down to 200 nm on a Lipex extruder (Northern Lipids, Burnaby, BC, Canada). FR-targeting PTX liposomes and FR-targeting PGD dendrosomes were purified by centrifugation at 1000 g for 30 min at room temperature. 2.3. Analysis of liposomal preparations 2.3.1. Size characterization Particle size of FR-targeting PTX liposomes or FR-targeting PGD dendrosomes were determined at 25  C by photon correlation spectroscopy (PCS) using Beckman Coulter Delsa Nano C instrumentation (Beckman Coulter, Brea, CA). Suspensions were diluted using sucrose-HEPES-saline buffer at 1:30 volume ratios. Samples were measured in triplicate and results reported as averages. 2.3.2. Morphological analysis The shape and surface morphology of FR-targeting PTX liposomes and FR-targeting PGD dendrosomes were observed via transmission electron microscopy (TEM). Liposomal suspensions were applied to carbon-coated copper grids, negatively stained by 2% uranyl acetate, and then air dried. TEM observation of the liposomes was carried out with a JEOL-100CX in the University of Texas Health Science Center at San Antonio (UTHSCSA) Pathology Electron Microscopy Lab.

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2.3.3. Quantitation of phospholipid content in the liposomes The PL concentration in the liposomes was determined using a previously described method [28]. Briefly, 2 ml of chloroform (Aldrich Chemical Co., Milwaulee, WI) and 2 ml of 0.1 M ammonium ferrothiocyanate (Aldrich) solution was added to 4 ml of undiluted liposomal suspension. Contents were shaken vigorously and centrifuged for 5 min at 300  g. The lower layer was collected and analyzed at 488 nm by spectrophotometry. Obtained values were compared to a standard curve to reveal PL concentration in liposomal suspensions. 2.3.4. Quantitation of the core content (PGD or PTX) in the liposomes The quantitation of the core content (PGD or PTX) was done by high-pressure liquid chromatography. The liposome sample was centrifuged at 1000 RCF for 25 min to allow for the sedimentation of non-encapsulated PGD or PTX, if any. Then, from the supernatant, 25 mL of the sample was removed to a new vial, followed by the addition of 50 mL of acetonitrile and vortex mixing. The mixture was then microfuged momentarily twice. An aliquot (20 mL) of the supernatant was injected on the HPLC (Milton Roy) and chromatographed isocratically through a 3.9 mm  300 mm, Waters mBondapak column packed with 5 m size, C18-derivatized silica particles, being eluted with a mixture of H2O/acetonitrile (55:45) at a flow rate of 1.5 mL/min at a pressure of about 3000 psi. The eluted components were monitored by a UV/vis monitor at 215 nm (AUFS 0.2) and were recorded on a linear chart recorder at the chart speed of 1 cm/min. The retention time and peak area of eluted components from liposome samples (the PGD dendrosome as well as the PTX liposome) were compared with those of the relative standard solutions. 2.3.5. Storage stability of liposomal preparations The liposomal preparations were stored at 4  C for up to 4 weeks. The stability of liposomal preparations was evaluated on Days 0, 7, 14, and 21. At each time point, the liposomal samples were centrifuged at 1000 RCF for 25 min to ensure that the leaked PGD or PTX content was sedimented. From each supernate, 25 mL aliquot was taken separately, mixed with 50 mL acetonitrile, microfuged and the PTX or PGD content was quantified for each time point (in triplicates) by HPLC, as above. 2.4. In vitro studies on PGD dendrosome and FR-targeting PTX liposomes 2.4.1. Cell cultures MDA-MB-231, MDA-MB-468, BT-20, and T47-D human breast cancer cells along with BUMPT mouse proximal tubule kidney cells were cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA). MDA-MB-435 human breast cancer cells and JEG-3 human choriocarcinoma cells were maintained in DMEM (Life Technologies). Both media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 1% Glutamax (Life Technologies) and 1% Antibiotic/Antimycotic solution (Life Technologies, 15240-062). All cell lines were maintained at 37  C in a humidified incubator in a 5% carbon dioxide atmosphere. 2.4.2. Western blot analysis Cellular lysates for Western blot analysis were prepared using previously described methodology [29]. Briefly, cells were washed three times with phosphate-buffered saline (PBS), treated with cold RIPA lysis buffer [50 mM TriseHCl, 150 mM NaCl, 0.5% Nonidet P40, 0.1 sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1 x protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN)

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and 1 mM sodium vanadate (Sigma)] on ice for 20 min, and harvested by scraping. Lysates were centrifuged at 14,000  g for 15 min at 4  C and supernatant was collected. Protein concentrations were determined using Micro-bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL) and proteins were stored at 80  C. Cell lysates containing equal amounts of protein (~20 mg) were electrophoresed on 8% SDS-polyacrylamide gels and transferred onto a 0.2 mm nitrocellulose membranes. Blots were probed with appropriate antibodies. Actin and folate receptor a (FRa) proteins were probed using anti-actin antibody produced in rabbit (Sigma) and anti-FRa monoclonal rabbit antibody (Sigma), respectively, as primary antibodies. After staining with appropriate secondary antibodies, blots were developed using enhanced chemiluminescence method. 2.4.3. Metabolic activity assay The effect of FR-targeting PGD dendrosomes and FR-targeting PTX liposomes on metabolic activity was determined by 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) assay (Roche Diagnostics) adapted from a similar previously-established protocol [24]. Cells (2  103 cells/ well) were seeded in 96-well culture plates and incubated for 24 h. Previous media was aspirated and replenished with drug treatments loaded in fresh folate-free RPMI 1640 (Life Technologies) culture medium supplemented with 5% FBS, 1% Glutamax and 1% Antibiotic/Antimycotic solution (Life Technologies, 15240-062) with or without 1 mM FA as a FR blocking agent. Treatments included blank (control), 5 nM, and 10 nM PTX as FR-targeting PTX liposomes or FR-targeting PGD dendrosomes. After 18 h incubation, cells were washed thrice with PBS and cultured in fresh media without drugs. Following an additional 54 h of incubation, 10 uL of WST-1 reagent was added to each well and plates were incubated for an additional 4 h. Absorbance of the converted WST-1 product was read at 415 nm on a microplate reader (Synergy 2, Biotek, Winooski, VT). 2.4.4. Apoptosis assay Annexin staining for measurement of apoptosis was performed using the FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, San Jose, CA). Briefly, MDA-MB-231 cells were seeded at 106 cells/dish and incubated for 24 h. The cells were then treated with 50 nM PTX as PTX liposome or PGD dendrosome. Media alone served as blank. After 48 h of treatment, cells were washed with cold PBS and resuspended in 100 ml binding buffer. Cells were stained with 5 ml of FITC Annexin V and 5 ml of propidium iodide (PI) for 15 min in the dark before diluting with an additional 400 ml binding buffer. Samples were analyzed by flow cytometry on a FACS Calibur (BD Biosciences, San Diego, CA). 2.5. In vivo antitumor efficacy All animal experiments were performed after obtaining the approval from the Institutional Animal Care and Use Committee of UTHSCSA, and animals were housed in accordance with the UTHSCSA institutional protocol for animal experiments. MDA-MB-231 cells were resuspended in 50% v/v serum-free culture medium/Matrigel Matrix (BD Biosciences, San Jose, CA). Approximately 2  106 MDA-MB-231 cells (100 ml) were injected into the mammary fatpad of 6 to 7-week-old female athymic nude mice. Once the mass of xenograft tumors reached an average volume of 90 mm3 (about 8 days after inoculation), mice were randomly assigned to three groups (n ¼ 7): control (saline vehicle), FR-targeting PTX liposomes (10 mg/kg PTX-equivalent), and FRtargeting PGD dendrosomes (10 mg/kg PTX-equivalent). Mice were given intraperitoneal injections every other day for a total of 3

injections. Two-dimensional tumor measurements were made weekly after the first dose. The tumor volume was calculated according to the following formula:

  Volume ¼ short diameter2  ðlong diameterÞ  0:5 The extent of tumor burden per animal was expressed in cubic millimeters. Mice were sacrificed 24 days after treatment initiation. 2.6. Statistical analysis Data are presented as the mean ± standard deviation (SD) unless otherwise indicated. One-way analysis of variance tests were used to investigate the differences between groups for apoptosis in vitro experiments and two-way analysis of variance tests for in vivo and metabolic activity in vitro experiments, followed by post hoc tests with Bonferroni correction for comparison between individual groups. Statistical significance was established at p < 0.05. Data for metabolic activity assays was investigated using Stata/IC 12.0 (StataCorp LP, College Station, TX). All other experiments were analyzed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). 3. Results and discussion Our earlier studies [18] showed that the PGD could actively target cathepsin B, an enzyme so often upregulated in breast cancer cells [30,31], to release the PTX based active ingredient. Compared to PTX alone, the PGD prodrug demonstrated increased cytotoxicity to breast cancer cells that had a moderate-to-high cathepsin B expression. The goal of this work, then, was to design an efficient drug delivery device that encapsulates PGD in its inner core so as to improve the therapeutic efficacy of the PTX active agent and to achieve increased targeting specificity, incorporating the elements of both passive and active targeting using two molecular differences between normal and cancer cells instead of just one targeting strategy. 3.1. Cancer cell targeting dendrosomes A cancer-specific cell surface receptor was chosen as an additional target for the nanotherapy. Cell surface receptors for FA are over expressed in large numbers in most cancer cells [32,33] but these receptors are only minimally distributed in normal tissues [34]; it is also a useful marker for cancer histopathologic identification [35,36]; FA is a high affinity ligand for FR, and it retains its receptor binding and endocytosis properties even if it is covalently linked to a wide variety of molecules. Therefore, FR serves as a functional cancer-specific receptor and FA has been generally employed as a ligand to target it [37]. Further, liposomes designed with a PEG-modified PL evade rapid clearance through RES, remain stabilized in blood circulation and improve bio-distribution and pharmacokinetics of the active ingredient [38]. Therefore, a folatebound and PEG-modified PL was synthesized for eventual incorporation in the proposed liposomal encapsulation of PGD (Fig. 2). 3.2. Phospholipid synthesis, liposome preparation and characterization FA-PEG-DSPE was prepared by the DCC facilitated acylation of amino-PEG-DSPE with FA (Scheme 1). The chemical identity of the product was confirmed by the combination of TLC analysis and the 1H NMR spectral analysis. The product chromatographed as a single spot on TLC, the Rf of which was different than that of all of the starting components of reaction; in the NMR spectrum, all 1H signals required for both folic acid amide as well as the PEG-DSPE

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Scheme 1. Synthesis of Folate bound poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE).

were accounted for. This FA-PEG-DSPE was included in the formulations of egg phosphatidylcholine liposomes, along with PEGDSPE to impart stealth capabilities to the PGD encapsulating device. The control samples were also created identically, using PTX instead of PGD as inner core component. In passive targeting, engineered particles exploit the EPR effect of leaky tumor vasculatures to favorably collect at tumor sites. Thus, the size of the engineered particles below the 600 nm level becomes a key factor in their success as drug delivery platforms for cancer therapy; the threshold vesicle size for extravasation into tumors has been recommended to be ~400 nm [39]. The liposomes

were extruded through a membrane pore size of 200 nm. The resultant liposomes were 161.3 nm ± 3.99 and 212.0 nm ± 5.14 for FR-targeting PTX and PGD liposomes, respectively, as determined by PCS. The morphology of the liposomes was characterized by transmission electron microscopy. Both FR-targeting PTX and PGD liposomes demonstrated discrete, spherical structures characteristic of vesicles (Fig. 3). Mean diameters were generally found in the 150e190 nm range for FR-targeting PTX liposomes and 190e220 range for FR-targeting PGD dendrosomes, affirming values obtained by PCS. These values demonstrate that the engineered particles were well below the upper limit of tumor vasculature pore sizes.

Fig. 3. Morphological shapes of (a) FR-targeting PTX liposomes and (b) FR-targeting PGD dendrosomes, as examined by TEM [Bar ¼ 100 nm].

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The PL content of the liposomes, as measured by the Stewart assay [28], was 17.3 mg/ml ± 2.1 and 18.3 mg/ml ± 0.9, respectively, for FR-targeting PTX and PGD liposomes. PTX was found to be encapsulated quantitatively in its liposomes (~100% ± 2.3%), while the loading efficiency PGD in dendrosome was 94% ± 4.8%. The stability of each drug in each liposomal formulation was monitored during storage for three weeks at 4  C (Fig. 4). FR-targeting PTX liposomes lost drug content relatively quickly, while the dendrosomes retained PGD without significant leakage. Thus, upon storage at 4  C, the retention of PTX in its liposomes was reduced to 22 ± 1.1%, 13 ± 0.8% and 10 ± 0.9% by the 1st, 2nd, and 3rd week, respectively. At the same temperature PGD was retained quantitatively in dendrosome till the end of 3-week storage. The improved drug retention during storage conditions using FRtargeting PGD dendrosomes represents a significant improvement over the PTX liposome formulation and has strong implications for pharmaceutical manufacturing and practical clinical utility. 3.3. In vitro studies on dendrosomes To correlate the effect of FR-targeting PGD dendrosomes on metabolic activity with the presence of FR, the levels of total FRa were investigated in an array of cells by Western Blot and enzymatic assays. Six breast cancer cell lines (MDA-MB-231, MDA-MB468, MDA-MB-435, BT-20, BT-549, and T47D) that demonstrate PTX sensitivity were chosen to represent the diseased state. Breast cancer cell lines were also selected for their diversity in specimen origin and cancer subtype to represent inherent breast cancer heterogeneity. Breast cancer with choriocarcinomatous features represents a distinct subset of the breast cancer spectrum, with 12e33% of female breast cancer patients expressing human chorionic gonadotropin (hCG) [40,41]. As such, the human choriocarcinoma cell line JEG-3 was chosen as a model to represent such pathological features. Finally, renal clearance is one of the primary methods of nanoparticle removal from the body. Given its large blood flow and ability to concentrate toxic compounds, the kidneys are also particularly sensitive to xenobiotics [42]. As such, BUMPT mouse kidney proximal tubule cells were chosen as a model for nanoparticle behavior against this critical normal tissue. In addition, kidney proximal tubule cells are reported to have a high folate receptor activity, and our previous research indicated a low

cathepsin B profile for the BUMPT cells, specifically [18]. Thus, the BUMPT cell line was thought to provide a way to possibly elucidate differences in single-level vs sequential active-targeting. The BUMPT cell line in particular is reported to behave similar to primary culture kidney proximal tubule cells in the conditions utilized here within [43,44], in contrast to the continuous E6/7 antigen expression of the alternative immortalized HK-2 cell line [45]. FRa was detected in all cells by Western blot (Fig. 5). However, marked differences were seen in its levels in the normal BUMPT mouse kidney proximal tubule cell line relative to that determined in the cancer cell lines (Fig. 5). Western blot analysis showed that FRa had the highest presence in MDA-MB-231, MDA-MB-468, and MDA-MB-435 cells. BT-20, JEG-3 and BT-549 had moderate levels of FRa, while BUMPT and T47D cells had the lowest levels of expression. A similar folate receptor profile was observed by receptor activity assay using calcein-loaded, FR-targeting liposomes (Supplemental Fig. 1). The effect of FR-targeting PGD dendrosomes on metabolic activity was studied using the panel of cell lines previously mentioned (Fig. 6). Metabolic activity assays, such as the WST-1 assay, have been previously established as indirect methods of assessing cytotoxic activity of drugs. FR-targeting PTX liposomes served as control, and cells treated with liposome or dendrosome in the presence of 1 mM FA as blocking agent served as a control to test FRa specificity. In general, the cancer cell lines demonstrated a stronger reduction in metabolic activity when treated with FRtargeting PGD dendrosomes than with FR-targeting PTX liposomes (Fig. 6). MDA-MB-435, JEG-3, and BT-20 cancer cell lines demonstrated statistically significant differences (p < 0.05) between FR-targeting PGD dendrosomes and FR-targeting PTX liposomes at both 5 nM and 10 nM concentrations of PTX. MDA-MB231 and MDA-MB-468 cells demonstrated statistically significant differences between FR-targeting PGD dendrosomes and FRtargeting PTX liposomes between 5 nM and 10 nM treatments respectively. Each of these cell lines had moderate-to-high levels of FRa expression and activity. Compared to untreated controls, MDAMB-231, MDA-MB-435, JEG-3, and BT-20 cell lines demonstrated statistically significant differences for PGD dendrosome treatments at both 5 and 10 nM, while MDA-MB-468 showed such a relationship for 10 nM PTX-equivalent PGD dendrosome treatment only. Among these five cell lines, only MDA-MB-231 at 10 nM

Percent Encapsulated Drug Content

Retention of Drugs in Liposomes 120 100 80 60

FR-targeting PTX Liposomes FR-targeting PGD Dendrosomes

40 20 0

0

1 2 Weeks of Incubation at 4°C

3

Fig. 4. Retention of encapsulated products from FR-targeting PGD dendrosomes and FR-targeting PTX liposomes. Both liposomal formulations were incubated at 4  C for three weeks. Results are reported as mean ± standard deviation of three independent experiments.

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a

FR α Actin JEG-3 | T47D | MB435 | BT20 | MB468 | BT549 | MB231 | BUMPT

Breast cancer cell lines

Normal kidney cell line

FRα Protein Expression (Western Blot) 1000 800 600 400 200 0

M D

JE G

-3 T4 7 A -M D B -4 35 B M T D -2 A 0 -M B -4 68 M BTD 54 A 9 -M B -2 31 B U M PT

b

Normalized FRα Expression

Choriocarcinoma cell line

Cell lines Fig. 5. (a) Western blot analysis of FRa in human breast cancer, human choriocarcinoma, and normal immortalized mouse kidney proximal tubule cells. Total cellular protein (20 mg) was electrophoresed on 8% SDS-PAGE gel and blotted onto a nitrocellulose membrane. FRa detection was performed by using anti-FRa monoclonal mouse antibody, staining with horseradish peroxidase-conjugated secondary antibody, and developing with enhanced chemiluminescence method. (b) Densitometric analysis of FRa western blot was performed using Adobe Photoshop software (Adobe Systems, San Jose, CA), normalized to actin intensity, and expressed relative to BUMPT values.

treatment and MDA-MB-435 at 5 nM and 10 nM treatments showed a statistically distinguishable result between untreated controls and FR-targeting PTX liposomes. Our previous experiments on the MDA-MB-231 cell line had indicated a relative IC50 of 3.29 nM for PTX and 0.640 nM PTXequivalent concentration for PGD [18]. Indeed, 5 nM and 10 nM PTX-equivalent concentrations were chosen as optimal to observe treatment effects. As compared to data from our previous experiments [18], the results of these experiments with MDA-MB-231, MDA-MB-468, MDA-MB-435, JEG-3, and BT-20 cell lines indicate that encapsulation of PGD and PTX within liposome architecture reduces cytotoxicity to even malignant cells. This corresponds to previously reported data that taxane-class drug encapsulation within liposome reduces cytotoxic activity [46,47]. However, delivering PTX through PGD dendrosome strongly recovers cytotoxic activity. When compared to our previously reported data [18], the cytotoxicity against cancer cells is often close to that achieved by PGD alone, which was our most successful treatment group previously [18]. To test for FRa specificity, cells were treated with 1 mM FA to block FR activity. Results showed that cytotoxicity caused by PGD dendrosome and FR-targeting PTX liposomes was FR-specific. In particular, MDA-MB-231, MDA-MB-468, MDA-MB-435, JEG-3, and BT-20 cells showed statistically significant differences between PGD dendrosome treatments with and without FA. Cells grown in presence of PGD dendrosome with FA showed a higher proliferation rate than without FA. This trend was mimicked by cells treated with FR-targeting PTX liposomes that had shown a response to the

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liposomes. As a control, we had also treated cells with 1 mM FA alone; results did not show an increase in metabolic activity due to FA alone (data not shown). Results from T47D and BT-549 breast cancer cells and BUMPT normal kidney cells presented insights into behavior towards normal cell lines with low target expression. Both T47D and BUMPT cells demonstrated low FR activity. In the case of T47D, only treatment with FR-targeting PTX liposomes at 10 nM concentration could demonstrate a statistically significant reduction in metabolic activity compared to untreated samples. For BUMPT normal kidney cells, treatments with FR-targeting PTX liposomes at 10 nM were significantly reduced (p < 0.05) from untreated controls as well as from their folate-blocked FR-targeting PTX liposomes counterparts. No other treatments that were compared for either of these cell lines could be distinguished from each other statistically. The results for BUMPT mouse normal kidney cells was anticipated given that, in our previous studies, we found these cells had low cathepsin B expression and weaker response to cathepsin B-targeting PGD dendrimer [18]. As a summary, our previous studies had also shown that BT-549 cells had low cathepsin B activity with concomitant lower sensitivity to PGD dendrimer, while MDA-MB-231, MDA-MB468, MDA-MB-435, BT-20, T47D, and JEG-3 cells had moderate-tohigh cathepsin B activity and increased PGD-induced cytotoxicity. When combined with low FR expression observed in this study, the lack of response to PGD dendrosome was apparent. T47D cancer cells, which had in our previous study demonstrated sensitivity to PGD dendrimer treatments due to its high cathepsin B expression, no longer showed reduced metabolic activity once PGD dendrimer was encapsulated in a folate-targeting liposome. BT-549 cancer cells, however, demonstrated a moderately-high level of FR activity, in line with that seen for MDA-MB-231 and MDA-MB-435 cells. Yet, the metabolic activity assay for BT-549 demonstrated similar results to that from T47D and BUMPT. Specifically, 10 nM FR-targeting PTX liposomes treatments showed statistically significant reductions in metabolic activity compared to untreated controls and folate-blocked counterparts. In addition, at 5 nM concentration, PTX liposome treatment showed reduction in metabolic activity that was statistically different from PGD dendrosome treatment, but statistically indistinguishable from untreated controls. Should the activity of PGD dendrosome have been dependent solely on outer core targeting of FR levels, the results from metabolic activity assays would have appeared similar to that of MDA-MB-435 cancer cells. Instead, it appears likely that inner core targeting of cellular cathepsin B also plays a role in determining cytotoxicity of FR-targeting PGD dendrosomes. Indeed, in our previous study [18], BT-549 was the only cancer cell line to demonstrate low cathepsin B activity and corresponding low sensitivity to PGD dendrimer. The results from this study indicate that encapsulating the PGD dendrimer in a folatetargeting liposome is not sufficient to recover lost cytotoxic activity as PTX is bound in prodrug form by an active-targeting peptide spacer. It should be noted that other factors also affect the intensity of response induced by the FR-targeting PGD dendrosomes, including cell line sensitivity to PTX. Indeed, such cell line variation may partially explain the null response observed for BUMPT cells at the 5 nM concentration level (Fig. 6h). This effect appears to be partially overcome by using a higher concentration, as seen in the results for the 10 nM PTX equivalent. Folate receptor binding and recycling has been observed to vary by cancer cell lines and normal tissue types [48]. Such variation in recycling behavior and binding kinetics may also explain the variation in cytotoxic response observed between cancer cell lines as well. These results were further reflected in a regression analysis comparing the magnitude of PGD dendrosome-induced cytotoxic

Fig. 6. Effect of FR-targeting PGD dendrosomes on metabolic activity. 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay of (a) MDAMB-231, (b) MDA-MB-468, (c) MDA-MB-435, (d) BT-20, T47D, (e)BT-549, (f) JEG-3, and (g) BUMPT cells were treated with 5 nM and 10 nM PTX delivered as FR-targeting PTX liposomes (PTX Liposome) or FR-targeting PGD dendrosomes (PGD Dendrosome) for 18 h and then allowed to grow in blank media for an additional 54 h. Folate-specific activity of liposomes and dendrosomes was tested by blocking FRa with 1 mM FA (PTX Liposome þ Fol and PGD Dendrosome þ Fol, respectively). Results are reported as mean ± standard deviation of three independent experiments (n ¼ 6). Statistically significant results are indicated by grouped lines (p < 0.05).

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Fig. 6. (continued).

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Fig. 6. (continued).

improvement at 10 nM treatment and an arbitrary variable representing combined cellular cathepsin B and FRa expression (Fig. 7). PGD dendrosome-induced mean metabolic activity was subtracted from PTX-induced mean metabolic activity for each cell line, resulting in a Metabolic Activity Differential that was plotted against the product of the cell line's cathepsin B [18] and FRa expression. The coefficient of determination r2 was 0.6223 at p ¼ 0.0200. This suggested a strong, statistically significant correlation between metabolic activity differential and combined cathepsin B-FRa expression. Thus, enhanced response to FRtargeting PGD dendrosomes over FR-targeting PTX liposomes is multifactorial but correlated with cathepsin B and FRa expression.

MDA-MB-231 breast cancer cells were chosen as model cells for further studies due to their favorable response rates in the metabolic activity assays, appropriateness as models of malignant metastatic breast cancer phenotype, tumorigenicity in vivo, and well-characterized nature with regards to PTX response [49,50]. To confirm if reduced metabolic activity by PGD conjugate was a result of cell death via the apoptotic pathway, annexin V-FITC staining was used to quantitatively determine the percentage of cells within a population that were undergoing apoptosis (Fig. 8). MDA-MB-231 cells were treated for 48 h with 50 nM PTX as PTX liposome or PGD dendrosome. Our results indicate that FRtargeting PGD dendrosomes elicited a strong response with an

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Metabolic Activity Differential (PTX Liposome-PGD Dendrosome) 0 -20 20 40 60

MDA-MB-435

y = -5.756495 + 0.0000824 x 2

r = 0.6223; p = 0.0200 JEG-3

MDA-MB-468

BT-20

MDA-MB-231

T47D BUMPT BT-549

0

200000 400000 600000 800000 Cathepsin B * Folate Receptor Expression (A.U.)

Fig. 7. Regression analysis between magnitude of FR-targeting PGD dendrosome-induced cytotoxic improvement (Metabolic Activity Differential) at 10 nM treatment and the product of cellular cathepsin B and FRa expression (Cathepsin B*Folate Receptor Expression). At a 10 nM PTX-equivalent treatment, FR-targeting PGD dendrosome-induced mean metabolic activity was subtracted from FR-targeting PTX liposome-induced mean metabolic activity for each cell line, resulting in a Metabolic Activity Differential. This was plotted against the product of the respective cell line's total cathepsin B expression [17] and folate receptor expression, conveyed in Arbitrary Units.

average of 83.8% of cells analyzed staining positively for annexin V. This was a statistically differentiable result compared to both untreated controls (23.6% annexin V positive) and PTX liposometreated samples (46.5%). The results suggested that cell death from the apoptotic pathway was a key component in the observed reduced proliferation rates in the WST-1 assays. Compared to our previous studies [18], the results also indicate that encapsulation of PTX in liposomes strongly reduces toxicity to cancer cells, but this toxicity is regained when PTX is delivered through PGD dendrosome form.

were 39% and 40% smaller than control tumor volumes for weeks-2 and-3, respectively. Tumor growth was slower in PGD dendrosometreated mice as compared to FR-targeting PTX liposomes-treated mice, resulting in statistically significant differences in tumor volume between the pair in weeks-2 and -3 after treatment initiation.

3.4. Antitumor effects of FR-targeting PGD dendrosomes on mice breast cancer xenografts While our studies confirmed the cytotoxic behavior of FRtargeting PGD dendrosomes towards cancer cells during in vitro testing, two-dimensional cell cultures cannot replicate the cellecell and cellematrix interactions of the inherently threedimensional system faced by drug delivery platforms in vivo, let alone the drastically different diffusion and transport properties [51]. The behavior of nanoparticles in vivo often differs significantly from the predicted behavior derived from in vitro cell culture studies [52]. As a result, to validate observations herewith and discern new insights, the effect of FR-targeting PGD dendrosomes as compared to FR-targeting PTX liposomes on tumor growth was evaluated through in vivo xenograft studies using MDA-MB-231 cells. Mice treated from all groups survived until pre-scheduled termination of experiment 32 days after tumor inoculation. No treatment showed external toxic or lethal sideeffects such as, furring, weakness, or fatigue. Treatment with FR-targeting PGD dendrosomes began to show reduced growth in tumor volume compared to control (saline injections) one week after treatment initiation and had statistically significant differences (p < 0.05) in tumor volume by weeks-2 and -3 (Fig. 9). PGD dendrosome-treated tumor volumes were up to 79% and 72% smaller than controls for weeks-2 and -3, respectively. Mice treated with FR-targeting PTX liposomes also demonstrated statistically significant differences in tumor volume for weeks-2 and -3 when compared to controls. PTX-treated tumor volumes

Fig. 8. PGD dendrosome exposure to MDA-MB-231 cells induces apoptosis. Annexin V staining was conducted with the use of a kit (Annexin V-FITC Apoptosis detection kit; BD Pharmingen) and analyzed by flow cytometry. MDA-MB-231 breast cancer cells were exposed to 50 nM PTX as FR-targeting PTX liposomes or FR-targeting PGD dendrosomes for 48 h. Untreated blank samples served as controls. Results are reported as mean ± standard deviation of three independent experiments. Statistically significant results are indicated by grouped lines (p < 0.05).

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For these respective weeks, PGD dendrosome-treated tumor volumes were 66% and 53% smaller than PTX liposome-treated tumor volumes. There was, therefore, a discernable impact from PGD dendrosome treatment on slowing the growth rate of MDA-MB231 tumor volumes as compared to PTX liposome treatment. It has been noted that FR-targeting vehicles may interact with both tumor cells and activated macrophages by a and b folate receptor isoforms, respectively [53]. Indeed, tumor associated macrophages have been a target of antitumor therapy [54]. While targeting both folate receptor isoforms may have a suppressive effect on tumor, it is difficult to elucidate the PGD dendrosomes' action on a specific isoform or both. As such, further experiments must be conducted to further determine the specific mechanism of action of antitumor activity of the PGD dendrosome in vivo.

array of cancer cells, when FR and cathepsin B activities were both at moderate-to-high levels. Lack of increased cytotoxicity by FRtargeting PGD dendrosomes, when either FR or cathepsin B targets were not upregulated, demonstrated the intended goal of sequential active-targeting. In vivo toxicity was similarly reflected in markedly higher tumor reduction as compared to traditional FRtargeting PTX liposome treatment. This research may eventually evolve a product platform with reduced systemic toxicity inherent with traditional chemotherapy and localized toxicity inherent to single-target nanoplatforms. The reduced side effects may allow for better tolerance of higher therapeutic load in advanced disease states and may increase patient lifespan. This research may lead to a new class of drug delivery device for other diseases also.

4. Conclusions

Acknowledgments

A novel, sequentially active-targeted drug delivery platform was synthesized by encapsulation of a cathepsin B-targeting PTXdendrimer conjugate in a FR-targeting liposome, and the device was characterized. Referred to as FR-targeting PGD dendrosomes, this delivery platform demonstrated better retention of PTX prodrug compared to traditional FR-targeting FR-targeting PTX liposomes, a potential boon for practical manufacturing and clinical concerns. Compared to these traditional liposomes, FR-targeting PGD dendrosomes also demonstrated increased cytotoxicity to an

Support for this research was provided, in part, by the Translational Science Training (TST) Across Disciplines program at the University of Texas Health Science Center at San Antonio, funded by the University of Texas System's Graduate Programs Initiative. The laboratory space and facilities were provided by the Department of Biomedical Engineering at the University of Texas at San Antonio, by the Department of Obstetrics and Gynecology at the University of Texas Health Science Center at San Antonio, and by RANN Research Corporation, San Antonio, Texas. We would also like to acknowledge the assistance and guidance provided by Dr. Manjeri Venkatachalam and Dr. Hui Geng, who supplied us with the BUMPT cell line and provided key information in understanding kidney proximal tubule cell line differences. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.03.039. References

Fig. 9. Effect of FR-targeting PGD dendrosomes on human breast cancer (MDA-MB231) xenograft tumors in mice. Subcutaneous tumors were generated by injecting 2  106 cells into the mammary fatpads of nude mice. When tumor volumes reached 90 mm3, therapy was initiated and administered every other day for three intraperitoneal injections total using the following treatments: control (saline), FR-targeting PTX liposomes, and FR-targeting PGD dendrosomes. Doses of 10 mg PTX/kg body weight were administered as FR-targeting PTX liposomes or FR-targeting PGD dendrosomes. Tumor volumes were measured 1, 2, and 3 weeks after therapy initiation and reported as mean ± standard error of the mean (n ¼ 7). Statistically significant results are indicated by grouped lines (p < 0.05). Results are reported as two different representations (a) and (b) of the same data set.

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