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Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting
COREL-08479; No of Pages 8 Journal of Controlled Release xxx (2016) xxx–xxx
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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting Rawan Omar, Yael Leichtman Bardoogo, Enav Corem-Salkmon, Boaz Mizrahi ⁎ Faculty of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel
a r t i c l e
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Article history: Received 10 July 2016 Received in revised form 19 September 2016 Accepted 21 September 2016 Available online xxxx Keywords: Drug delivery Self-assembly Conjugates Camptothecin Cancer
a b s t r a c t Camptothecin (CPT) is a naturally occurring cytotoxic alkaloid having a broad spectrum of antitumor activity. Unfortunately, it has low bioavailability and encapsulation efficiency, limiting its clinical use. We report on our efforts to develop a novel drug delivery prototype composed of a short, star hydrophilic polyethylene glycol (PEG) backbone and hydrophobic CPT (PEG4-CPT). The amphiphilic bio-conjugate self-assembles in water into stable spherical nano-particles with a mean diameter of 200 nm and CPT substitution percentage of 27%w/w. CPT is released in a sustained release profile without burst effect. In addition, PEG4-CPT nano-particles are able to load a co-drug, water soluble or non-water soluble doxorubicin and release them simultaneously with the free CPT. The biological evaluation of PEG4-CPT against HeLa cells showed improved cellular uptake and enhanced cytotoxicity compared to free CPT. Thus, in this approach CPT acts in two ways: As the hydrophobic segment that enables self-assembly in water and as a potent anticancer agent. This concept of combining hydrophobic drugs and short star polymers shows great potential for efficient delivery of hydrophobic chemotrophic drugs as well as for drugs with inherent stability and pharmacokinetic barriers. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Over the last three decades, there has been a significant advance in drug-delivery research that has allowed optimal concentrations of drugs for longer periods with minimal toxic effects [1]. A major application of controlled delivery systems has been the delivery of anticancer drugs, given that many possess high lipophilicity and low water solubility [2], in addition to severe toxicity [3]. The use of commonly available delivery systems in cancer treatment, however, is still limited by two main concerns [4]. First, instability of the carrier and unfavorable release kinetics including significant burst release and loss of drug portion before it reaches the tumor site have been found to compromise clinical efficacy [5]. Second, inefficient encapsulation of the drug meaning that more carrier materials and excipients are used, so that the potential for long-term accumulation and the risk of toxicity and immunogenicity is increased [6], thus limiting these systems' applicability. New concepts that offer viable solutions for hydrophobic molecules with inherent stability and toxicity are greatly needed [7,8]. Several approaches have been utilized for the delivery of hydrophobic drugs, among the most promising are: (1) A self-assembly approach, where the components are composed of hydrophilic and hydrophobic parts that spontaneously self-assemble into defined structures in aqueous media [9]. This approach have some drawbacks: Micelles, for example, suffer from ⁎ Corresponding author. E-mail address: [email protected] (B. Mizrahi).
intrinsically poor chemical stability and pre-mature disintegration in the circulation, limiting their clinical use [10,11]. (2) A bio-conjugation approach, where a drug is attached to a high molecular weight watersoluble backbone such as polyethylene glycol (PEG) [12] or a polysaccharide [13]. Alternatively, the hydrophobic drug can be conjugated to an amphiphilic copolymer through chemical conjugation, followed by self-assembling the drug-conjugated polymers into stable micelles [14]. This latter approach have been found to have several advantages over traditional free drug administration including increased aqueous solubility of hydrophobic drugs while reducing their side effects, increased stability and improved intravascular half-life [15]. Nevertheless, this approach often increases the potential for long-term accumulation and frequently suffers from low drug loading [6]. Here we propose a method that would combine the advantages of self-assembly and the bio-conjugate approaches (Fig. 1). We hypothesized that a delivery system composed of hydrophobic drug molecules attached to a short multi-armed hydrophilic core in a labile fashion could have a potential as drug delivery system due to its high number of active end groups per polymer unit and reported ability to selfassemble into stable particles of the desired size [16]. To produce such a drug delivery system, we designed a conjugate composed of starshaped low molecular weight PEG4 (2000 Da), the hydrophilic segment, and the anti-cancer drug CPT, the hydrophobic segment. CPT, a natural cytotoxic quinolone alkaloid [17] was chosen as a model drug due to its broad although very potent in vitro spectrum against a broad range of solid tumors (e.g., primary and metastatic
http://dx.doi.org/10.1016/j.jconrel.2016.09.025 0168-3659/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 1. Synthesis scheme of PEG4–CPT and concept of suggested delivery system. Low Mw multi-armed PEG (the hydrophilic segment) is conjugated to 2 or 3 hydrophobic drugs (blue) via a labile bond. Thus, the drug works in two ways: through the hydrophobic segment that enables self-assembly and through its pharmacological activity. The bio-conjugate self-assembles into nanostructures with enhanced drug solubility, stability and an improved pharmacokinetic profile. Upon drug release via a labile linkage, the structure is disassembled and the low Mw polymer is secreted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
colon carcinoma, small cell lung carcinoma, ovarian, breast, pancreatic, and stomach cancers [18]). It is clinically ineffective due to its low bioavailability, low water solubility, and severe side effects after injection. In addition, CPT is unstable in physiological conditions where its lactone ring readily opens, yielding the inactive carboxylate form of the drug [19]. 2. Materials and methods 2.1. Materials (S)-(+)-Camptothecin was purchased from Chem-Impex International Inc. (IL, USA). Four-armed polyethylene glycol-2000 Da (PEG4, 2000 Da, each arm comprising 500 Da) was purchased from JenKem Technology Co. Ltd. (Beijing, China). Nile red, thionyl chloride, succinic anhydride, Dulbecco's phosphate-buffered saline (PBS) pH 7.4, Dulbecco's modified Eagle's medium (DMEM), normal donkey serum, propidium iodide (PI) and esterase from porcine liver were purchased from Sigma Aldrich (MO, USA). 4-Dimethylaminopyridine, 99% (DMAP) was purchased from Alfa Aesar (ward Hill, MA, USA). Penicillin-streptomycin, fetal bovine serum (FBS) and L-glutamine were purchased from Biological Industries (Israel). Paraformaldehyde was purchased from Electron Microscopy Sciences (PA, USA). A CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit was purchased from Promega (WI, USA). Fluoromount G was purchased from SouthernBiotech (AL, USA). Cathepsin D antibody was purchased from R&D Systems (Minneapolis, MN, USA). All solvents were of analytical grade, purchased from Bio-Lab Ltd. (Israel). 2.2. Synthesis of PEG4-CPT conjugates 2.2.1. Synthesis of PEG4-COOH Synthesis of PEG4-COOH was conducted according to Atassi et al. [20] (Fig. 1). PEG4 (4 g, 2 mmol) was dissolved in anhydrous pyridine (25 ml) and heated to 50 °C. Succinic anhydride (8 g, 80 mmol) was added over a period of several hours and the mixture was stirred for
an additional 2 h. The pyridine was evaporated under a vacuum, and the residue was dissolved in double distilled water (DDW). The remaining reactants were removed by dialysis against DDW (MWCO 1000 Da). Finally, samples were lyophilized to gain the final product. 2.2.2. Synthesis of PEG4-CPT Thionyl chloride (80 μl, 1.1 mmol) in 10 ml anhydrous dichloromethane (DCM) was added in a dropwise manner to PEG4–COOH (0.25 g, 0.115 mmol) dissolved in 5 ml of DCM (Fig. 1). The mixture was allowed to stir for 24 h at 60 °C and thionyl chloride excess was removed under reduced pressure to obtain PEG4–COCl. To obtain PEG4CPT, CPT (0.348 g, 1 mmol), suspended in iced cold anhydrous chloroform (10 ml), was mixed with DMAP (0.094 g, 0.77 mmol) and an anhydrous chloroform solution of PEG4-COCl in a dropwise manner. The mixture was allowed to stir at room temperature for 24 h. Unreacted CPT was removed by vacuum filtration (Whatman™ Filter paper, grade 1, 11 μm) and the final product was obtained through precipitation into an ice-cold diethyl ether and dried under a vacuum. 2.3. Characterization of PEG4-COOH and PEG4-CPT 2.3.1. Chemical characterization The absorption spectroscopy of PEG4-COOH, PEG4-CPT and pure CPT in DMSO were measured by a UV/Vis Spectrophotometer (Ultraspec 2100 pro, Amersham Pharmacia Biotech, UK). 1H–NMR data of the chemical structures were obtained using a Bruker Avance III 400 MHz NMR spectrometer (MA, USA) in D2O or CDCl3. The molecular weight of PEG4 before and after conjugation was evaluated with gel permeation chromatography (GPC) (Viscotek VE 1122, Malvern Instruments, UK) with PSS GRAM 1000 Å + PSS GRAM 30 Å columns (PSS, Germany) in Tetrahydrofuran (THF). 2.3.2. Physical characterization Size distribution and stability of the nano-particles were measured with a NanoSight NS300 instrument at 532 nm laser beam (Malvern Instruments, UK). PEG4-CPT solutions were dispersed in PBS pH 7.4 (10
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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mg/ml) and diluted according to the manufacturer's instructions. Data were analyzed with NanoSight NTA 3.1 software. The morphology of the nano-particles was defined using Tecnai T12G2 transmission electron microscopes (TEM, FEI). A 400-mesh carbon-coated grid was placed on a 20 μl sample drop for 2 min and blotted with a filter paper. The sample was chemically stained by placing the grid on a 20 μl drop of 1% uranyl acetate for 2 min followed by blotting with a filter paper and air-drying. Specimens for TEM were examined in a Philips CM120 transmission electron microscope operating at 120 kV. Images were recorded digitally on Gatan MultiScan 791 camera using the DigitalMicrograph software (Gatan, U.K.) [21,22]. Further examination was conducted using Cryo-TEM (FEI Tecnai Spirit G12, acceleration voltage 120 kV, Gatan CCD camera 1 × 1,Gatan Cryo transfer holder 626). The critical micelle concentration (CMC) of the PEG4-CPT conjugates was evaluated by fluorescence detection (Varioskan Flash Multimode Reader, Thermo Scientific, US). An aqueous solution of the nanoparticles was prepared and diluted to various concentrations with DDW. Nile red (4 μl, 0.97 mg/ml in acetone) was added to each sample (200 μl) and the CMC was evaluated by measuring the emission spectrum at 570 b λ b 750 nm with 550 nm excitation light [23]. Results are the average of four independent repetitions (n = 4). 2.4. In vitro release of CPT The release of CPT from PEG4-CPT nano-particles was conducted in dialysis membrane tubes (Midi GeBaFlex-tube, MWCO = 1 kD, Gene Bio-Application Ltd., Israel) against PBS pH 7.4, containing 1% DMSO at 37 °C with constant shaking at 60 RPM. 350 μl of PEG4-CPT (in PBS) were placed in each tube (n = 4). For comparison purposes, pure CPT in 350 μl DMSO was also measured using similar conditions. CPT release from PEG4-CPT was also evaluated with the presence of 72 U of esterase in PBS incubated at the same conditions. The releasing media was sampled at predetermined intervals and was replaced by fresh media. The concentrations of CPT in collected release media were evaluated using a fluorimeter at excitation of 369 nm and emission of 437 nm. The release of CPT from PEG4-CPT was also conducted with the presence of 72 U of esterase. Release studies were limited to 180 h since the apparent terminal elimination half-life (t1/2) of free CPT after administration of PEG-CPT conjugates was estimated to be 77.46 h (±36 h) [24]. 2.5. Formation of Dox free base Doxorubicin (Dox) free base was prepared by alkaline precipitation of Dox hydrochloride followed by its filtration. Dox·HCl (0.2 g, 0.34 mmol) was dissolved in 12 ml DDW and the pH was adjusted to 8 by adding a 10% NaOH solution. The precipitate was collected by centrifugation, washed several times with DDW and lyophilized. 2.6. Encapsulation and release of DOX·HCl and Dox free base Dox·HCl or Dox free base (2.77 mg, 0.005 mmol) and 4 mg PEG4-CPT were dissolved in 2 ml DMSO and stirred in the dark for 2 h. The solution was added dropwise to 10 ml DDW while stirring. Entrapped Dox was collected by centrifugation using Amicon ultra centrifugal filters with 100 K cutoff (Merck Millipore Ltd., Ireland) at 14,000 g (4 °C) for 30 min. Drug loading (DL) and encapsulation efficiency (EE) of DOX were calculated by comparing the fluorescence emission of DOX at 560 nm with 480 nm excitation against a calibration curve of DOX in DMSO and using the following equations: Drug loading ð%w=wÞ ¼
mdrug loaded in particles 100% mPEG4−CPT
Encapsulation efficiency ð%w=wÞ ¼
mdrug loaded in particles 100% mtotal drug
3
Where mdrug loaded in particles is the mass of the drug entrapped in the PEG4-CPT nano-particles, mPEG4 −CPT is the mass of the PEG4-CPT nanoparticles, and mtotal drug is the mass of the feeding drug. 2.7. Cell uptake, cytotoxicity and immunofluorescence 2.7.1. Cell uptake Cell uptake and localization of intracellular CPT were assessed on human epitheloid cervix carcinoma cells (HeLa). Cells were cultured in a 24-well plate at a density of 4 · 104 cells/well, 24 h prior to treatment, at 37 °C in DMEM supplemented with 10% FBS, 1% penicillinstreptomycin and 1% glutamine. Cultures were maintained in a 95% air/5% carbon dioxide atmosphere, at 95% relative humidity. Cells were incubated for indicated time periods with PEG4-CPT nanoparticles (1 mg/ml). After fixation with 4% paraformaldehyde, cells were washed with PBS, and permeabilized with 0.1% triton-x-100 in PBS for 10 min at RT. Blocking was carried out with 10% normal donkey serum for 30 min in PBS containing 0.1% BSA. Cells were incubated for 3 h with goat anti-human cathepsin D antibody, as a lysosome marker, diluted 1:400 in PBS containing 0.1% BSA. After washing the unbonded antibody, cells were incubated for 45 min at RT with secondary antibody, Alexa donkey anti-goat 488, diluted 1:1000 in PBS containing 0.1% BSA. Cells were washed with PBS, and for nucleus staining, propidium iodide (PI) 1 mg/ml, diluted 1:750 in DDW, was added to each well, and incubated for 15 min. Then cells were rewashed with PBS. An immuno-stained coverslip was taken out of each well, and placed on a microslide containing fluoromount G. Cells exposed to PEG4 only (at the same weight ratio) and cells with no treatment served as controls. The intracellular localization of CPT was observed using a Zeiss LSM 700 laser scanning confocal microscope with a Plan Apochromat 63/ 1.4 NA oil DIC lens. Samples were scanned with three different lasers: 405 nm (for CPT visualization), 488 nm (for lysosomes visualization), and 555 nm (for PI visualization). 2.7.2. Cytotoxicity of PEG4-CPT HeLa cells were seeded in 24-well plate at a density of 7 · 104 cells/ well, incubated as mentioned above for 24 h with 0.001 to 1 mg/ml of PEG4-CPT in DMEM. For comparison purposes, equivalent concentrations of CPT were incubated as well. Cell viability was evaluated by MTS assay using a CellTiter 96® solution according to the manufacturer's protocol, relatively to unexposed cells. Results are averages of 4 replications. 2.7.3. Localization of PEG4-CPT Cellular uptake of CPT in HeLa cells was also observed with 1 mg/ml PEG4-CPT nano-particles after 1 h treatment. Cells were cultured in a 24-well plate with a density of 5 · 104 cells/well, 24 h prior to treatment, and incubated as mentioned above. Cells were incubated for 1 h with PEG4-CPT vesicles (1 mg/ml) in complete medium. They were then washed with PBS and fixed with 4% paraformaldehyde. After fixation, cells were rewashed with PBS. PI (1 mg/ml), diluted 1:1000 in DDW was added to each well and incubated for 15 min. Cells were rewashed with PBS. Cells exposed to PEG4 only (at the same weight ratio) and cells with no treatment served as controls. Acquisition and analysis was carried out using ZEN software. Of note, in order to gain better analysis of the results, CPT color was digitally changed to red and the nucleus color was digitally changed to blue using ZEN software. 3. Results and discussion 3.1. Synthesis and chemical characterization of PEG4-CPT PEG4-CPT was synthesized by the formation of PEG4-COOH, followed by conjugating with CPT by ester bonds (Fig. 1). Synthesis of the intermediate PEG4-COOH and the final PEG4-CPT product were compared to pure PEG4 by 1H–NMR (Fig. S1). While PEG4-COOH showed a new
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 2. (A) The absorption spectroscopy of PEG4-COOH, CPT and PEG4-CPT; (B) GPC analysis of PEG4 and the synthesized PEG4-CPT.
peak at 2.6 ppm, attributed to the COOH group [25], PEG4-CPT showed new peaks above 7.5 ppm, attributed to CPT [26]. Substitution percentage, as determined by comparing the integration value of PEG4 (indicated by a in Fig. S2) to the integration value of the carboxylic acid proton (indicated by b in Fig. S2), was around 100%. For PEG4-CPT (Fig. S3), new peaks in the range between 7.5 and 8.5 ppm (indicated by c-e) can be attributed to the hydrogen atoms on the A and B rings of CPT [26]. Formation of PEG4-CPT through PEG4-COOH was also confirmed by UV-VIS (Fig. 2A). CPT presents maximum peaks around 260, 296 and 365– 383 nm, in agreement with the literature data [27]. Compared to PEG4-COOH, the PEG4-CPT spectrum presents new peaks at 296 nm and above 365 nm, attributable to the CPT molecule. Substitution percentage, measured by UV–VIS, was found to be 50% (27%w/w), comparable to efficiencies reported for modification of CPT with other short yet linear PEGs [28]. Of note, our previous reports of substituting small molecules with star PEGs of similar Mw showed substitution rates of around 77% [29,30].This variance from our current result can be attributed to the rigid planar hydrophobic nature of the CPT that limits accessibility to binding sites [31]. The analysis molecular weight of pure PEG4 and PEG4-CPT was obtained by GPC (Fig. 2B). The molecular weight of PEG4-CPT was found to be 2154 Da whereas the molecular weight of the pure PEG4 was 1402 Da. These results verify the success of the synthesis and the notion that each PEG4 molecule was replaced by 2–3 CPT molecules. Overall, 1 H–NMR, UV–Vis and GPC results confirmed the successful synthesis of PEG4-CPT and the high substitution percentage.
3.2. Physical characterization We next investigated how PEG4-CPT forms nano-structures in aqueous solution. CMC was measured by a spectrophotometric technique using Nile red as the hydrophobic probe. Nile red is poorly watersoluble unless a hydrophobic compartment is provided. As a result, the CMC value was determined by plotting a graph of the maximum peak wavelength as a function of PEG4-CPT concentrations (Fig. 3A). The CMC of PEG4-CPT was calculated to be 0.7 mg/ml, implying that assemblies of the PEG4-CPT are formed above this concentration. This CMC value is up to 10 fold greater compared with other, linear PEGCPT conjugates. For example, CPT-SS-PEG2000-SS-CPT conjugate [32] showed CMC value of 0.06 mg/ml while a series of mPEGylated α, β -poly (L-aspartic acid)-CPT conjugates had values between 0.01 and 0.5 mg/ml [33]. The greater CMC value of the PEG4-CPT system can be explained by the decreased hydrophobic/hydrophilic block ratio compared to the linear system being investigated [34]. Transmission electron microscopy (TEM) confirmed that PEG4-CPT form discrete spherical aggregates in the range of 100–200 nm (insets of Fig. 3B, C and S4). TEM analysis showed spherical aggregates with condensed hydrophobic core and a brighter hydrophilic shell. Similar self-assembly behavior of four-armed PEG into spherical aggregates was reported by us [29] and by others [35]. Nanoparticle tracking analysis (NTA), enabling accurate sizing and a clear distinction of the various sizes with excellent peak resolution [36], clearly revealed formation of micellar structures within the range of 50–
Fig. 3. (A) The critical micelle concentration (CMC) graph, and emission spectra of Nile red in the presence of different concentrations of PEG4-CPT (inset); (B) Size distribution and morphology of PEG4-CPT in PBS, pH = 7.4. Size distribution of PEG4-CPT nano-particles immediately after formation (red) and after 48 h (blue), as determined by NTA and TEM analysis (inset); (C) TEM image of PEG4-CPT nano-particle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. The release experiment. (A) The release of free CPT, CPT from PEG4-CPT (with esterase), and CPT from PEG4–CPT (without esterase); (B) The release of CPT and Dox·HCl from PEG4CPT; (C) The release of CPT and DOX Free Base from PEG4-CPT.
250 nm (Fig. 3B, mean diameter 171.9 ± 7.5 nm). Particles remained in the same size range (mean diameter 135.3 ± 8.1 nm) after 48 h at 37 °C, suggesting aqueous stability. Noteworthy is that considering that our system is designed to target cancer tissue, the formed nano-particles were found to be of the appropriate size so as to take advantage of the enhanced permeability and retention (EPR) effect [32]. 3.3. In vitro release of CPT To assess the delivery performance of PEG4-CPT, CPT release patterns were studied in vitro in PBS, pH 7.4 at 37 °C by measuring the fluorescence emission of CPT at 437 nm (Fig. 4A). For comparison purposes, the release of CPT in the presence of esterase was also evaluated (Fig. 4A). In the case of free CPT, about 90% of the total drug was measured within 24 h. Of note, the relative slow release of the free CPT is attributed to the sharp decrease in the concentration of DMSO, from 100 to 1% with the introduction of the aqueous media. From the PEG4-CPT conjugate, however, only 26% was released during the same period without a burst release phenomenon. After four days, 43% of the loaded CPT was released from the nano-particles. The introduction of esterase increased the release rate by about 25%, probably through triggering the breaking of the ester bonds. The usefulness of the PEG4-CPT system as a platform to carry an additional drug was demonstrated by its containment and then release of doxorubicin HCl (Fig. 4B) or doxorubicin free base (Fig. 4C). DOX·HCl (DL = 4.34%w/w, EE = 6.20%w/w in Table 1) was released within 24 h, suggesting that the water-soluble form of the drug easily diffuses out through the CPT membrane. On the other hand, the poor watersoluble Dox free base (DL = 4.23%w/w, EE = 6.04%w/w in Table 1) was released in a controlled fashion over several days, indicating the incorporation of the drug inside the hydrophobic bilayer (inset of Fig. 4B and C). Thus, these experiments demonstrate that both water-soluble and poor water-soluble drugs can be encapsulated in the PEG4-CPT nanostructures and released as co-drugs in a controlled fashion. Table 1 Calculations of drug loading (DL) and encapsulation efficiency (EE) of CPT, DOX·HCl and Dox free base.
Conjugation CPT Loading DOX·HCl Dox free base
Drug loading (% w/w)
Encapsulation efficiency (% w/w)
27
–
4.34 4.23
6.20 6.04
Interestingly, the release kinetics of CPT was increased in the presence of Dox·HCl. A similar trend was observed with free base Dox: Over 90% of the total CPT were released within 24 h compared to b 50% in the absent of Dox. The greater release of CPT from the PEG4-CPT can be explained by the negative effect of Dox on the stability of the nanoparticles thus exposing the ester bonds to hydrolysis and enabling faster CPT release [37]. Nevertheless, from a clinical point of view, exploiting the hydrophobic or the hydrophilic compartment to deliver a second drug may be very promising, since combinatorial treatments are used in many diseases including in several types of cancer [38]. 3.4. Cell penetration and cytotoxicity of PEG4-CPT The cellular uptake of PEG4-CPT nano-particles was demonstrated using a laser scanning confocal microscope (Fig. 5A). HeLa cells were incubated with PEG4-CPT nano-particles for 1 h and the intracellular localization of CPT was evaluated based on its fluorescence properties [39]. A clear blue fluorescence signal, attributed to CPT, was detected, indicating successful penetration into the cell. In contrast, PEG4 (without CPT) of similar concentration did not reveal any fluorescence signal, verifying our results. We note that our attempts to introduce free CPT were not successful since DMSO, required to dissolve the hydrophobic CPT, either did not prevent aggregation of CPT when introduced into the aqueous cell media or caused deformation of the cell membrane. These attempts further indicate the usefulness of our system for the delivery of poor water-soluble drugs and their targeted delivery into the cell. Next, the cytotoxicity effect of PEG4-CPT conjugates on HeLa cells was evaluated by MTS assay (Fig. 5B). Viability (as percentage relative to cells not exposed to PEG4-CPT or free CPT) showed a dosedependent behavior for both free CPT and PEG4-CPT nano-particles. PEG4-CPT nano-particles showed similar or slightly higher cytotoxicity compared to free CPT. The higher level of cytotoxicity of the conjugated CPT can, in part, be explained by the stability of CPT. The lactone ring of free CPT tends to open under physiological pH and yields an inactive carboxylate form [40]. Researchers, however, have reported that conjugation of CPT at the 20th position stabilizes the molecular structure of CPT, and therefore, maintains its activity [41]. Moreover, conjugation of hydrophobic drugs such as CPT to a water-soluble polymeric platform was reported to enhance cellular uptake by endocytic pathway [41]. The fact that the endocytic pathway mechanism is involved was evident by the accumulation of CPT in the lysosome of HeLa cells (Fig. 6). The localization of CPT in the lysosome was observed within 1 h of incubation (yellow spots), which emphasizes the entrance of CPT into
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 5. Cells experiments. (A) Cellular uptake of 1 mg/ml of PEG4-CPT in HeLa cells after 1 h of incubation, PEG4 (1 mg/ml) and untreated HeLa cells are used as the control; (B) HeLa cells' viability (relative to unexposed HeLa cells) using an MTS assay, 22 h after exposure to PEG4-CPT nano-particles and to free drug.
the nucleus through the lysosome. Though some CPT was observed outside the lysosomes (red spots in Fig. 6), these may have been ones that had not as yet entered the lysosomes or had already escaped within the 1 h incubation period. After 24 h, most fluorescence signaling of CPT was observed at the nucleus of the cells and some in the cytosol, suggesting that CPT had escaped from the lysosomes compartments. These results further suggest that the PEG4-CPT approach can potentially serve in the anti-cancer formulation of hydrophobic drugs, where elevated drug concentration in the cytoplasm and nucleus is desired [42].
drug, sustained release profile, and an ability to carry both water soluble and poor water soluble co-drugs while offering efficient cell uptake with minimal toxicity. Thus, the drug acts in two ways: by enabling selfassembly in water (the hydrophobic segment) and as a pharmacologic anticancer agent. Since the covalent binding between the drug and the polymer is labile under physiological conditions, upon drug release the amphiphilic structure is destroyed and the short polymer may easily be cleared. Further experimental research including in vivo studies are required in order to confirm our observations.
4. Conclusions
Acknowledgments
We developed a new family of star shaped bio-conjugates, having a short hydrophilic backbone and hydrophobic therapeutic agents. The amphiphilic bio-conjugate self-assembles in water to form nanostructures with excellent properties: very high loading of the hydrophobic
We thank Professor Dganit Danino and Dr. Inbal Abutbul-Ionita for the TEM analysis. We also thank the Russell Berrie department for granting a scholarship for excellent students in Nanoscience and Nanotechnology research.
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 6. Cellular uptake of PEG4-CPT (1 mg/ml) and localization in the lysosome after 1 and 24 h incubation.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.09.025.
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Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting Rawan Omar, Yael Leichtman Bardoogo, Enav Corem-Salkmon, Boaz Mizrahi ⁎ Faculty of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel
a r t i c l e
i n f o
Article history: Received 10 July 2016 Received in revised form 19 September 2016 Accepted 21 September 2016 Available online xxxx Keywords: Drug delivery Self-assembly Conjugates Camptothecin Cancer
a b s t r a c t Camptothecin (CPT) is a naturally occurring cytotoxic alkaloid having a broad spectrum of antitumor activity. Unfortunately, it has low bioavailability and encapsulation efficiency, limiting its clinical use. We report on our efforts to develop a novel drug delivery prototype composed of a short, star hydrophilic polyethylene glycol (PEG) backbone and hydrophobic CPT (PEG4-CPT). The amphiphilic bio-conjugate self-assembles in water into stable spherical nano-particles with a mean diameter of 200 nm and CPT substitution percentage of 27%w/w. CPT is released in a sustained release profile without burst effect. In addition, PEG4-CPT nano-particles are able to load a co-drug, water soluble or non-water soluble doxorubicin and release them simultaneously with the free CPT. The biological evaluation of PEG4-CPT against HeLa cells showed improved cellular uptake and enhanced cytotoxicity compared to free CPT. Thus, in this approach CPT acts in two ways: As the hydrophobic segment that enables self-assembly in water and as a potent anticancer agent. This concept of combining hydrophobic drugs and short star polymers shows great potential for efficient delivery of hydrophobic chemotrophic drugs as well as for drugs with inherent stability and pharmacokinetic barriers. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Over the last three decades, there has been a significant advance in drug-delivery research that has allowed optimal concentrations of drugs for longer periods with minimal toxic effects [1]. A major application of controlled delivery systems has been the delivery of anticancer drugs, given that many possess high lipophilicity and low water solubility [2], in addition to severe toxicity [3]. The use of commonly available delivery systems in cancer treatment, however, is still limited by two main concerns [4]. First, instability of the carrier and unfavorable release kinetics including significant burst release and loss of drug portion before it reaches the tumor site have been found to compromise clinical efficacy [5]. Second, inefficient encapsulation of the drug meaning that more carrier materials and excipients are used, so that the potential for long-term accumulation and the risk of toxicity and immunogenicity is increased [6], thus limiting these systems' applicability. New concepts that offer viable solutions for hydrophobic molecules with inherent stability and toxicity are greatly needed [7,8]. Several approaches have been utilized for the delivery of hydrophobic drugs, among the most promising are: (1) A self-assembly approach, where the components are composed of hydrophilic and hydrophobic parts that spontaneously self-assemble into defined structures in aqueous media [9]. This approach have some drawbacks: Micelles, for example, suffer from ⁎ Corresponding author. E-mail address: [email protected] (B. Mizrahi).
intrinsically poor chemical stability and pre-mature disintegration in the circulation, limiting their clinical use [10,11]. (2) A bio-conjugation approach, where a drug is attached to a high molecular weight watersoluble backbone such as polyethylene glycol (PEG) [12] or a polysaccharide [13]. Alternatively, the hydrophobic drug can be conjugated to an amphiphilic copolymer through chemical conjugation, followed by self-assembling the drug-conjugated polymers into stable micelles [14]. This latter approach have been found to have several advantages over traditional free drug administration including increased aqueous solubility of hydrophobic drugs while reducing their side effects, increased stability and improved intravascular half-life [15]. Nevertheless, this approach often increases the potential for long-term accumulation and frequently suffers from low drug loading [6]. Here we propose a method that would combine the advantages of self-assembly and the bio-conjugate approaches (Fig. 1). We hypothesized that a delivery system composed of hydrophobic drug molecules attached to a short multi-armed hydrophilic core in a labile fashion could have a potential as drug delivery system due to its high number of active end groups per polymer unit and reported ability to selfassemble into stable particles of the desired size [16]. To produce such a drug delivery system, we designed a conjugate composed of starshaped low molecular weight PEG4 (2000 Da), the hydrophilic segment, and the anti-cancer drug CPT, the hydrophobic segment. CPT, a natural cytotoxic quinolone alkaloid [17] was chosen as a model drug due to its broad although very potent in vitro spectrum against a broad range of solid tumors (e.g., primary and metastatic
http://dx.doi.org/10.1016/j.jconrel.2016.09.025 0168-3659/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 1. Synthesis scheme of PEG4–CPT and concept of suggested delivery system. Low Mw multi-armed PEG (the hydrophilic segment) is conjugated to 2 or 3 hydrophobic drugs (blue) via a labile bond. Thus, the drug works in two ways: through the hydrophobic segment that enables self-assembly and through its pharmacological activity. The bio-conjugate self-assembles into nanostructures with enhanced drug solubility, stability and an improved pharmacokinetic profile. Upon drug release via a labile linkage, the structure is disassembled and the low Mw polymer is secreted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
colon carcinoma, small cell lung carcinoma, ovarian, breast, pancreatic, and stomach cancers [18]). It is clinically ineffective due to its low bioavailability, low water solubility, and severe side effects after injection. In addition, CPT is unstable in physiological conditions where its lactone ring readily opens, yielding the inactive carboxylate form of the drug [19]. 2. Materials and methods 2.1. Materials (S)-(+)-Camptothecin was purchased from Chem-Impex International Inc. (IL, USA). Four-armed polyethylene glycol-2000 Da (PEG4, 2000 Da, each arm comprising 500 Da) was purchased from JenKem Technology Co. Ltd. (Beijing, China). Nile red, thionyl chloride, succinic anhydride, Dulbecco's phosphate-buffered saline (PBS) pH 7.4, Dulbecco's modified Eagle's medium (DMEM), normal donkey serum, propidium iodide (PI) and esterase from porcine liver were purchased from Sigma Aldrich (MO, USA). 4-Dimethylaminopyridine, 99% (DMAP) was purchased from Alfa Aesar (ward Hill, MA, USA). Penicillin-streptomycin, fetal bovine serum (FBS) and L-glutamine were purchased from Biological Industries (Israel). Paraformaldehyde was purchased from Electron Microscopy Sciences (PA, USA). A CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit was purchased from Promega (WI, USA). Fluoromount G was purchased from SouthernBiotech (AL, USA). Cathepsin D antibody was purchased from R&D Systems (Minneapolis, MN, USA). All solvents were of analytical grade, purchased from Bio-Lab Ltd. (Israel). 2.2. Synthesis of PEG4-CPT conjugates 2.2.1. Synthesis of PEG4-COOH Synthesis of PEG4-COOH was conducted according to Atassi et al. [20] (Fig. 1). PEG4 (4 g, 2 mmol) was dissolved in anhydrous pyridine (25 ml) and heated to 50 °C. Succinic anhydride (8 g, 80 mmol) was added over a period of several hours and the mixture was stirred for
an additional 2 h. The pyridine was evaporated under a vacuum, and the residue was dissolved in double distilled water (DDW). The remaining reactants were removed by dialysis against DDW (MWCO 1000 Da). Finally, samples were lyophilized to gain the final product. 2.2.2. Synthesis of PEG4-CPT Thionyl chloride (80 μl, 1.1 mmol) in 10 ml anhydrous dichloromethane (DCM) was added in a dropwise manner to PEG4–COOH (0.25 g, 0.115 mmol) dissolved in 5 ml of DCM (Fig. 1). The mixture was allowed to stir for 24 h at 60 °C and thionyl chloride excess was removed under reduced pressure to obtain PEG4–COCl. To obtain PEG4CPT, CPT (0.348 g, 1 mmol), suspended in iced cold anhydrous chloroform (10 ml), was mixed with DMAP (0.094 g, 0.77 mmol) and an anhydrous chloroform solution of PEG4-COCl in a dropwise manner. The mixture was allowed to stir at room temperature for 24 h. Unreacted CPT was removed by vacuum filtration (Whatman™ Filter paper, grade 1, 11 μm) and the final product was obtained through precipitation into an ice-cold diethyl ether and dried under a vacuum. 2.3. Characterization of PEG4-COOH and PEG4-CPT 2.3.1. Chemical characterization The absorption spectroscopy of PEG4-COOH, PEG4-CPT and pure CPT in DMSO were measured by a UV/Vis Spectrophotometer (Ultraspec 2100 pro, Amersham Pharmacia Biotech, UK). 1H–NMR data of the chemical structures were obtained using a Bruker Avance III 400 MHz NMR spectrometer (MA, USA) in D2O or CDCl3. The molecular weight of PEG4 before and after conjugation was evaluated with gel permeation chromatography (GPC) (Viscotek VE 1122, Malvern Instruments, UK) with PSS GRAM 1000 Å + PSS GRAM 30 Å columns (PSS, Germany) in Tetrahydrofuran (THF). 2.3.2. Physical characterization Size distribution and stability of the nano-particles were measured with a NanoSight NS300 instrument at 532 nm laser beam (Malvern Instruments, UK). PEG4-CPT solutions were dispersed in PBS pH 7.4 (10
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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mg/ml) and diluted according to the manufacturer's instructions. Data were analyzed with NanoSight NTA 3.1 software. The morphology of the nano-particles was defined using Tecnai T12G2 transmission electron microscopes (TEM, FEI). A 400-mesh carbon-coated grid was placed on a 20 μl sample drop for 2 min and blotted with a filter paper. The sample was chemically stained by placing the grid on a 20 μl drop of 1% uranyl acetate for 2 min followed by blotting with a filter paper and air-drying. Specimens for TEM were examined in a Philips CM120 transmission electron microscope operating at 120 kV. Images were recorded digitally on Gatan MultiScan 791 camera using the DigitalMicrograph software (Gatan, U.K.) [21,22]. Further examination was conducted using Cryo-TEM (FEI Tecnai Spirit G12, acceleration voltage 120 kV, Gatan CCD camera 1 × 1,Gatan Cryo transfer holder 626). The critical micelle concentration (CMC) of the PEG4-CPT conjugates was evaluated by fluorescence detection (Varioskan Flash Multimode Reader, Thermo Scientific, US). An aqueous solution of the nanoparticles was prepared and diluted to various concentrations with DDW. Nile red (4 μl, 0.97 mg/ml in acetone) was added to each sample (200 μl) and the CMC was evaluated by measuring the emission spectrum at 570 b λ b 750 nm with 550 nm excitation light [23]. Results are the average of four independent repetitions (n = 4). 2.4. In vitro release of CPT The release of CPT from PEG4-CPT nano-particles was conducted in dialysis membrane tubes (Midi GeBaFlex-tube, MWCO = 1 kD, Gene Bio-Application Ltd., Israel) against PBS pH 7.4, containing 1% DMSO at 37 °C with constant shaking at 60 RPM. 350 μl of PEG4-CPT (in PBS) were placed in each tube (n = 4). For comparison purposes, pure CPT in 350 μl DMSO was also measured using similar conditions. CPT release from PEG4-CPT was also evaluated with the presence of 72 U of esterase in PBS incubated at the same conditions. The releasing media was sampled at predetermined intervals and was replaced by fresh media. The concentrations of CPT in collected release media were evaluated using a fluorimeter at excitation of 369 nm and emission of 437 nm. The release of CPT from PEG4-CPT was also conducted with the presence of 72 U of esterase. Release studies were limited to 180 h since the apparent terminal elimination half-life (t1/2) of free CPT after administration of PEG-CPT conjugates was estimated to be 77.46 h (±36 h) [24]. 2.5. Formation of Dox free base Doxorubicin (Dox) free base was prepared by alkaline precipitation of Dox hydrochloride followed by its filtration. Dox·HCl (0.2 g, 0.34 mmol) was dissolved in 12 ml DDW and the pH was adjusted to 8 by adding a 10% NaOH solution. The precipitate was collected by centrifugation, washed several times with DDW and lyophilized. 2.6. Encapsulation and release of DOX·HCl and Dox free base Dox·HCl or Dox free base (2.77 mg, 0.005 mmol) and 4 mg PEG4-CPT were dissolved in 2 ml DMSO and stirred in the dark for 2 h. The solution was added dropwise to 10 ml DDW while stirring. Entrapped Dox was collected by centrifugation using Amicon ultra centrifugal filters with 100 K cutoff (Merck Millipore Ltd., Ireland) at 14,000 g (4 °C) for 30 min. Drug loading (DL) and encapsulation efficiency (EE) of DOX were calculated by comparing the fluorescence emission of DOX at 560 nm with 480 nm excitation against a calibration curve of DOX in DMSO and using the following equations: Drug loading ð%w=wÞ ¼
mdrug loaded in particles 100% mPEG4−CPT
Encapsulation efficiency ð%w=wÞ ¼
mdrug loaded in particles 100% mtotal drug
3
Where mdrug loaded in particles is the mass of the drug entrapped in the PEG4-CPT nano-particles, mPEG4 −CPT is the mass of the PEG4-CPT nanoparticles, and mtotal drug is the mass of the feeding drug. 2.7. Cell uptake, cytotoxicity and immunofluorescence 2.7.1. Cell uptake Cell uptake and localization of intracellular CPT were assessed on human epitheloid cervix carcinoma cells (HeLa). Cells were cultured in a 24-well plate at a density of 4 · 104 cells/well, 24 h prior to treatment, at 37 °C in DMEM supplemented with 10% FBS, 1% penicillinstreptomycin and 1% glutamine. Cultures were maintained in a 95% air/5% carbon dioxide atmosphere, at 95% relative humidity. Cells were incubated for indicated time periods with PEG4-CPT nanoparticles (1 mg/ml). After fixation with 4% paraformaldehyde, cells were washed with PBS, and permeabilized with 0.1% triton-x-100 in PBS for 10 min at RT. Blocking was carried out with 10% normal donkey serum for 30 min in PBS containing 0.1% BSA. Cells were incubated for 3 h with goat anti-human cathepsin D antibody, as a lysosome marker, diluted 1:400 in PBS containing 0.1% BSA. After washing the unbonded antibody, cells were incubated for 45 min at RT with secondary antibody, Alexa donkey anti-goat 488, diluted 1:1000 in PBS containing 0.1% BSA. Cells were washed with PBS, and for nucleus staining, propidium iodide (PI) 1 mg/ml, diluted 1:750 in DDW, was added to each well, and incubated for 15 min. Then cells were rewashed with PBS. An immuno-stained coverslip was taken out of each well, and placed on a microslide containing fluoromount G. Cells exposed to PEG4 only (at the same weight ratio) and cells with no treatment served as controls. The intracellular localization of CPT was observed using a Zeiss LSM 700 laser scanning confocal microscope with a Plan Apochromat 63/ 1.4 NA oil DIC lens. Samples were scanned with three different lasers: 405 nm (for CPT visualization), 488 nm (for lysosomes visualization), and 555 nm (for PI visualization). 2.7.2. Cytotoxicity of PEG4-CPT HeLa cells were seeded in 24-well plate at a density of 7 · 104 cells/ well, incubated as mentioned above for 24 h with 0.001 to 1 mg/ml of PEG4-CPT in DMEM. For comparison purposes, equivalent concentrations of CPT were incubated as well. Cell viability was evaluated by MTS assay using a CellTiter 96® solution according to the manufacturer's protocol, relatively to unexposed cells. Results are averages of 4 replications. 2.7.3. Localization of PEG4-CPT Cellular uptake of CPT in HeLa cells was also observed with 1 mg/ml PEG4-CPT nano-particles after 1 h treatment. Cells were cultured in a 24-well plate with a density of 5 · 104 cells/well, 24 h prior to treatment, and incubated as mentioned above. Cells were incubated for 1 h with PEG4-CPT vesicles (1 mg/ml) in complete medium. They were then washed with PBS and fixed with 4% paraformaldehyde. After fixation, cells were rewashed with PBS. PI (1 mg/ml), diluted 1:1000 in DDW was added to each well and incubated for 15 min. Cells were rewashed with PBS. Cells exposed to PEG4 only (at the same weight ratio) and cells with no treatment served as controls. Acquisition and analysis was carried out using ZEN software. Of note, in order to gain better analysis of the results, CPT color was digitally changed to red and the nucleus color was digitally changed to blue using ZEN software. 3. Results and discussion 3.1. Synthesis and chemical characterization of PEG4-CPT PEG4-CPT was synthesized by the formation of PEG4-COOH, followed by conjugating with CPT by ester bonds (Fig. 1). Synthesis of the intermediate PEG4-COOH and the final PEG4-CPT product were compared to pure PEG4 by 1H–NMR (Fig. S1). While PEG4-COOH showed a new
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 2. (A) The absorption spectroscopy of PEG4-COOH, CPT and PEG4-CPT; (B) GPC analysis of PEG4 and the synthesized PEG4-CPT.
peak at 2.6 ppm, attributed to the COOH group [25], PEG4-CPT showed new peaks above 7.5 ppm, attributed to CPT [26]. Substitution percentage, as determined by comparing the integration value of PEG4 (indicated by a in Fig. S2) to the integration value of the carboxylic acid proton (indicated by b in Fig. S2), was around 100%. For PEG4-CPT (Fig. S3), new peaks in the range between 7.5 and 8.5 ppm (indicated by c-e) can be attributed to the hydrogen atoms on the A and B rings of CPT [26]. Formation of PEG4-CPT through PEG4-COOH was also confirmed by UV-VIS (Fig. 2A). CPT presents maximum peaks around 260, 296 and 365– 383 nm, in agreement with the literature data [27]. Compared to PEG4-COOH, the PEG4-CPT spectrum presents new peaks at 296 nm and above 365 nm, attributable to the CPT molecule. Substitution percentage, measured by UV–VIS, was found to be 50% (27%w/w), comparable to efficiencies reported for modification of CPT with other short yet linear PEGs [28]. Of note, our previous reports of substituting small molecules with star PEGs of similar Mw showed substitution rates of around 77% [29,30].This variance from our current result can be attributed to the rigid planar hydrophobic nature of the CPT that limits accessibility to binding sites [31]. The analysis molecular weight of pure PEG4 and PEG4-CPT was obtained by GPC (Fig. 2B). The molecular weight of PEG4-CPT was found to be 2154 Da whereas the molecular weight of the pure PEG4 was 1402 Da. These results verify the success of the synthesis and the notion that each PEG4 molecule was replaced by 2–3 CPT molecules. Overall, 1 H–NMR, UV–Vis and GPC results confirmed the successful synthesis of PEG4-CPT and the high substitution percentage.
3.2. Physical characterization We next investigated how PEG4-CPT forms nano-structures in aqueous solution. CMC was measured by a spectrophotometric technique using Nile red as the hydrophobic probe. Nile red is poorly watersoluble unless a hydrophobic compartment is provided. As a result, the CMC value was determined by plotting a graph of the maximum peak wavelength as a function of PEG4-CPT concentrations (Fig. 3A). The CMC of PEG4-CPT was calculated to be 0.7 mg/ml, implying that assemblies of the PEG4-CPT are formed above this concentration. This CMC value is up to 10 fold greater compared with other, linear PEGCPT conjugates. For example, CPT-SS-PEG2000-SS-CPT conjugate [32] showed CMC value of 0.06 mg/ml while a series of mPEGylated α, β -poly (L-aspartic acid)-CPT conjugates had values between 0.01 and 0.5 mg/ml [33]. The greater CMC value of the PEG4-CPT system can be explained by the decreased hydrophobic/hydrophilic block ratio compared to the linear system being investigated [34]. Transmission electron microscopy (TEM) confirmed that PEG4-CPT form discrete spherical aggregates in the range of 100–200 nm (insets of Fig. 3B, C and S4). TEM analysis showed spherical aggregates with condensed hydrophobic core and a brighter hydrophilic shell. Similar self-assembly behavior of four-armed PEG into spherical aggregates was reported by us [29] and by others [35]. Nanoparticle tracking analysis (NTA), enabling accurate sizing and a clear distinction of the various sizes with excellent peak resolution [36], clearly revealed formation of micellar structures within the range of 50–
Fig. 3. (A) The critical micelle concentration (CMC) graph, and emission spectra of Nile red in the presence of different concentrations of PEG4-CPT (inset); (B) Size distribution and morphology of PEG4-CPT in PBS, pH = 7.4. Size distribution of PEG4-CPT nano-particles immediately after formation (red) and after 48 h (blue), as determined by NTA and TEM analysis (inset); (C) TEM image of PEG4-CPT nano-particle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 4. The release experiment. (A) The release of free CPT, CPT from PEG4-CPT (with esterase), and CPT from PEG4–CPT (without esterase); (B) The release of CPT and Dox·HCl from PEG4CPT; (C) The release of CPT and DOX Free Base from PEG4-CPT.
250 nm (Fig. 3B, mean diameter 171.9 ± 7.5 nm). Particles remained in the same size range (mean diameter 135.3 ± 8.1 nm) after 48 h at 37 °C, suggesting aqueous stability. Noteworthy is that considering that our system is designed to target cancer tissue, the formed nano-particles were found to be of the appropriate size so as to take advantage of the enhanced permeability and retention (EPR) effect [32]. 3.3. In vitro release of CPT To assess the delivery performance of PEG4-CPT, CPT release patterns were studied in vitro in PBS, pH 7.4 at 37 °C by measuring the fluorescence emission of CPT at 437 nm (Fig. 4A). For comparison purposes, the release of CPT in the presence of esterase was also evaluated (Fig. 4A). In the case of free CPT, about 90% of the total drug was measured within 24 h. Of note, the relative slow release of the free CPT is attributed to the sharp decrease in the concentration of DMSO, from 100 to 1% with the introduction of the aqueous media. From the PEG4-CPT conjugate, however, only 26% was released during the same period without a burst release phenomenon. After four days, 43% of the loaded CPT was released from the nano-particles. The introduction of esterase increased the release rate by about 25%, probably through triggering the breaking of the ester bonds. The usefulness of the PEG4-CPT system as a platform to carry an additional drug was demonstrated by its containment and then release of doxorubicin HCl (Fig. 4B) or doxorubicin free base (Fig. 4C). DOX·HCl (DL = 4.34%w/w, EE = 6.20%w/w in Table 1) was released within 24 h, suggesting that the water-soluble form of the drug easily diffuses out through the CPT membrane. On the other hand, the poor watersoluble Dox free base (DL = 4.23%w/w, EE = 6.04%w/w in Table 1) was released in a controlled fashion over several days, indicating the incorporation of the drug inside the hydrophobic bilayer (inset of Fig. 4B and C). Thus, these experiments demonstrate that both water-soluble and poor water-soluble drugs can be encapsulated in the PEG4-CPT nanostructures and released as co-drugs in a controlled fashion. Table 1 Calculations of drug loading (DL) and encapsulation efficiency (EE) of CPT, DOX·HCl and Dox free base.
Conjugation CPT Loading DOX·HCl Dox free base
Drug loading (% w/w)
Encapsulation efficiency (% w/w)
27
–
4.34 4.23
6.20 6.04
Interestingly, the release kinetics of CPT was increased in the presence of Dox·HCl. A similar trend was observed with free base Dox: Over 90% of the total CPT were released within 24 h compared to b 50% in the absent of Dox. The greater release of CPT from the PEG4-CPT can be explained by the negative effect of Dox on the stability of the nanoparticles thus exposing the ester bonds to hydrolysis and enabling faster CPT release [37]. Nevertheless, from a clinical point of view, exploiting the hydrophobic or the hydrophilic compartment to deliver a second drug may be very promising, since combinatorial treatments are used in many diseases including in several types of cancer [38]. 3.4. Cell penetration and cytotoxicity of PEG4-CPT The cellular uptake of PEG4-CPT nano-particles was demonstrated using a laser scanning confocal microscope (Fig. 5A). HeLa cells were incubated with PEG4-CPT nano-particles for 1 h and the intracellular localization of CPT was evaluated based on its fluorescence properties [39]. A clear blue fluorescence signal, attributed to CPT, was detected, indicating successful penetration into the cell. In contrast, PEG4 (without CPT) of similar concentration did not reveal any fluorescence signal, verifying our results. We note that our attempts to introduce free CPT were not successful since DMSO, required to dissolve the hydrophobic CPT, either did not prevent aggregation of CPT when introduced into the aqueous cell media or caused deformation of the cell membrane. These attempts further indicate the usefulness of our system for the delivery of poor water-soluble drugs and their targeted delivery into the cell. Next, the cytotoxicity effect of PEG4-CPT conjugates on HeLa cells was evaluated by MTS assay (Fig. 5B). Viability (as percentage relative to cells not exposed to PEG4-CPT or free CPT) showed a dosedependent behavior for both free CPT and PEG4-CPT nano-particles. PEG4-CPT nano-particles showed similar or slightly higher cytotoxicity compared to free CPT. The higher level of cytotoxicity of the conjugated CPT can, in part, be explained by the stability of CPT. The lactone ring of free CPT tends to open under physiological pH and yields an inactive carboxylate form [40]. Researchers, however, have reported that conjugation of CPT at the 20th position stabilizes the molecular structure of CPT, and therefore, maintains its activity [41]. Moreover, conjugation of hydrophobic drugs such as CPT to a water-soluble polymeric platform was reported to enhance cellular uptake by endocytic pathway [41]. The fact that the endocytic pathway mechanism is involved was evident by the accumulation of CPT in the lysosome of HeLa cells (Fig. 6). The localization of CPT in the lysosome was observed within 1 h of incubation (yellow spots), which emphasizes the entrance of CPT into
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 5. Cells experiments. (A) Cellular uptake of 1 mg/ml of PEG4-CPT in HeLa cells after 1 h of incubation, PEG4 (1 mg/ml) and untreated HeLa cells are used as the control; (B) HeLa cells' viability (relative to unexposed HeLa cells) using an MTS assay, 22 h after exposure to PEG4-CPT nano-particles and to free drug.
the nucleus through the lysosome. Though some CPT was observed outside the lysosomes (red spots in Fig. 6), these may have been ones that had not as yet entered the lysosomes or had already escaped within the 1 h incubation period. After 24 h, most fluorescence signaling of CPT was observed at the nucleus of the cells and some in the cytosol, suggesting that CPT had escaped from the lysosomes compartments. These results further suggest that the PEG4-CPT approach can potentially serve in the anti-cancer formulation of hydrophobic drugs, where elevated drug concentration in the cytoplasm and nucleus is desired [42].
drug, sustained release profile, and an ability to carry both water soluble and poor water soluble co-drugs while offering efficient cell uptake with minimal toxicity. Thus, the drug acts in two ways: by enabling selfassembly in water (the hydrophobic segment) and as a pharmacologic anticancer agent. Since the covalent binding between the drug and the polymer is labile under physiological conditions, upon drug release the amphiphilic structure is destroyed and the short polymer may easily be cleared. Further experimental research including in vivo studies are required in order to confirm our observations.
4. Conclusions
Acknowledgments
We developed a new family of star shaped bio-conjugates, having a short hydrophilic backbone and hydrophobic therapeutic agents. The amphiphilic bio-conjugate self-assembles in water to form nanostructures with excellent properties: very high loading of the hydrophobic
We thank Professor Dganit Danino and Dr. Inbal Abutbul-Ionita for the TEM analysis. We also thank the Russell Berrie department for granting a scholarship for excellent students in Nanoscience and Nanotechnology research.
Please cite this article as: R. Omar, et al., Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting, J. Control. Release (2016), http://dx.doi.org/10.1016/j.jconrel.2016.09.025
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Fig. 6. Cellular uptake of PEG4-CPT (1 mg/ml) and localization in the lysosome after 1 and 24 h incubation.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.09.025.
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