A novel rapamycin-polymer conjugate based on a new poly(ethylene glycol) multiblock copolymer

Abstract

Purpose

Rapamycin has demonstrated potent anti-tumor activity in preclinical and clinical studies. However, the clinical development of its formulations was hampered due to its poor solubility and undesirable distribution in vivo. Chemical modification of rapamycin presents an opportunity for overcoming the obstacles and improving its therapeutic index. The objective of this study is to develop a drug-polymer conjugate to increase the solubility and cellular uptake of rapamycin.

Methods

We developed the rapamycin-polymer conjugate using a novel, linear, poly(ethylene glycol) (PEG) based multiblock copolymer. Cytotoxicity and cellular uptake of the rapamycin-polymer conjugate were evaluated in various cancer cells.

Results

The rapamycin-polymer conjugate provides enhanced solubility in water compared with free rapamycin and shows profound activity against a panel of human cancer cell lines. The rapamycin-polymer conjugate also presents high drug loading capacity (wt% ~ 26%) when GlyGlyGly is used as a linker. Cellular uptake of the conjugate was confirmed by confocal microscopy examination of PC-3 cells that were cultured in the presence of FITC-labled polymer (FITC-polymer).

Conclusion

This study suggests that the rapamycin-polymer conjugate is a novel anti-cancer agent that may provide an attractive strategy for treatment of a wide variety of tumors.

INTRODUCTION

Rapamycin, also known as sirolimus, is a carboxylic macrolide compound that was originally discovered as the product of a bacteria from soil sample of island Rapa Nui in the South Pacific [1]. It was first developed as an antifungal drug by Ayerst Research Laboratories, but the potent immunosuppressive and anti-proliferative effects were discovered later [2]. Rapamycin was approved as an immunosuppressive agent by the US Food and Drug Administration (FDA) in 1999. Due to its unique cytostatic effect, rapamycin has also entered into clinical trials to treat various cancers, including breast cancer, prostate cancer and glioblastoma [3-5]. The molecular mechanism of rapamycin is to inhibit the mammalian target of rapamycin (mTOR) signaling pathway by directly binding to FK-binding protein 12 (FKBP12) and mTOR1. Because of the importance of the mTOR signal pathway in cancer development, rapamcyin was found to be active against almost all types of solid tumors [2]. Rapamycin exhibits an exquisite selectivity to mTOR and demonstrates potent anti-tumor activity in the nanomolar range in vitro. Besides inhibiting cancer cell proliferation, rapamycin can also induce tumor endothelial cell death and vessel thrombosis by acting as a vascular AKT/mTOR inhibitor, which facilitates its anti-tumor effects [6-7].

Although rapamycin has broad and potent anti-tumor activity, it is insoluble in water and possesses poor distribution in vivo, which dramatically limits its clinical application [8-10]. Structurally, rapamycin does not contain any acidic or basic group that can increase aqueous solubility by salt formation [8, 11]. As a result, rapamycin is very hydrophobic and the solubility in water is only about 33 μg/mL (2.6 μg/mL in some reports) [12]. Moreover, it is very difficult to prepare injectable rapamycin formulations for preclinical/clinical studies because rapamycin is only slightly soluble in many cosolvents such as ethanol, PEG 400 and polysorbate 80 [8]. Rapamycin also exhibits a poor distribution profile in vivo. Due to the ubiquitous presence of the FK-binding proteins (high affinity to rapamycin) in red blood cells (RBCs), rapamycin in the blood exhibits a preferential distribution in RBCs. As reported by Yatscoff et al., 95% of rapamycin in the blood is distributed in RBCs compared to 3.1% in the plasma, 1.0% in lymphocytes and 1.0% in granulocytes [9]. Because RBCs can protect rapamycin from hepatic metabolism and renal filtration, rapamycin exhibits a relatively long terminal half-life (61-72 hr in humans) compared with other small molecules [13]. However, low partition in the plasma dramatically hinders the diffusion of rapamycin from the blood into tumor cells. In addition, it was reported that rapamycin was extensively distributed among many tissues in rats (tissue to blood partition coefficient Kp > 40 in some cases) because of its lipophilic nature [14]. The extensive distribution of rapamcyin in tissues is believed to account for its high apparent distribution volume (5.6-17.6 L/kg) and toxic effects [15-16].

To overcome these limitations, rapamycin has been chemically modified to hydrophilic analogues, such as CCI-779, RAD001 and AP23573 (Figure 1A) [17]. CCI-779, also known as Temsirolimus (Torisel, Wyeth), is an ester prodrug of rapamycin for treatment of renal cell carcinoma. By introducing 2,2-bis(hydroxylmethyl) propionic ester at the 42-OH of rapamycin, CCI-779 increases its hydrophilicity and makes the analogue readily soluble for intravenous formulation [18]. RAD001, also known as Everolimus (Afinitor, Novartis), is a hydroxyl-ethyl ether of rapamycin with improved aqueous solubility and oral absorption [19]. AP23573 (Deforolimus, Merck/Ariad) is a novel non-prodrug rapamycin analogue in which 42-OH is conjugated with a phosphate group to increase aqueous solubility and oral bioavailability [20]. However, these rapamycin analogues (rapalogues) still have limited aqueous solubility (CCI-779: 0.12 mg/mL; RAD001: 0.1 mg/mL; AP23573: < 1 mg/mL) [15]. Cosolvent such as ethanol is always required for intravenous formulation [21]. Moreover, it was reported that the rapalogue CCI-779 could be quickly hydrolyzed into rapamycin by plasma esterase, which would redistribute into RBCs. Although other two rapalogues, RAD001 and AP23573, cannot be converted into rapamycin in vivo, moderate binding to RBCs was still observed in preclinical studies [22]. Therefore, it is necessary to explore other rapamycin conjugates to improve aqueous solubility and tissue distribution profile, especially to minimize the distribution to RBCs.

Figure 1.

Engineering of the rapamycin-polymer conjugate. (A). Structural comparison of rapamycin and rapalogues (CCI-779, AP23573 and RAD001). All of them are chemically modified at 42-OH position which is believed to minimally compromise their bioactivities. (B). The schematic graph illustrating the architecture of rapamycin-polymer conjugate 8c. Instead of small molecular-weight moieties (highlighted in red in rapalogues structure), rapamycin in 8c is conjugated to high molecular-weight polymer which can dramatically change its solubility and biodistribution profile. Rapamycin (Rapa) is uniformly distributed throughout the multiblock copolymer. The drug molecules are separated from each other by PEG to avoid aggregation; (C). The representative chemical structure of one block of the polymer-drug conjugate.

Compared to the chemical modification, direct conjugation of rapamycin to a polymer may offer a better approach to overcome its limitations. Many hydrophobic drugs, such as paclitaxel and camptothecin, have demonstrated excellent aqueous solubility and pharamcokinetics after being conjugated to hydrophilic polymers [23-26]. However, rapamycin is sensitive to light, temperature and pH, which make it hard to tolerate many chemical reactions. Although rapamycin has been successfully conjugated with wortmanin and other small molecules under mild reaction conditions, little attempt has been made to conjugate it with macromolecules, such as polymers and antibodies [27-29]. In this study, we developed a novel, water-soluble, poly(ethylene glycol) (PEG) based multiblock copolymer for rapamycin conjugation. To conjugate rapamycin with the polymer, a tri-peptide linker GlyGlyGly was conjugated with rapamycin at 42-OH and then grafted onto the pendant carboxylic groups in polymer chains. The rapamycin-polymer conjugate exhibits excellent solubility in water and potent anti-tumor activity, suggesting its promising potential for clinical development.

EXPERIMENTAL SECTION

Materials

All reagents listed below were obtained from commercial sources and used without further purification. Rapamycin was purchased from LC laboratories (Woburn, MA). NHSPEG₃₄₀₀-NHS was purchased from Nanocs Inc. (New York, NJ). 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-Hydroxybenzotriazole (HOBT) were ordered from Genscript Inc. (Piscataway, NJ). Thiazolyl Blue was obtained from RPI Corp. (Prospect, IL). Fmoc-Lys(Boc)-OH and Trt-Gly-OH were purchased from Chem-Impex International Inc. (Wood Dale, IL). TO-PRO-3 stain was obtained from Life Technologies (Grand Island, NY). All solvents, including anhydrous solvents, HPLC grade solvents and other common organic solvents were purchased from commercial suppliers and used without further purification.

Cell Culture

All the cell lines used in this study were obtained from ATCC. LNCaP, PC-3, MCF-7, MCF-7/HER2 and SK-BR-3 cells were grown in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. HeLa and DU145 cells were cultured in DMEM medium supplemented with 10% FBS and 100 units/mL penicillin-100 μg/mL streptomycin mixtures. All the cells were grown at 37 °C in a humidified atmosphere with 5% CO₂. Media were changed every other day, and the cells were passaged when they reached 80-90 % confluency.

Bis(ε-amino-L-Lysine Benzyl ester) Glutaric amide, bis(ε-Lys-OBn)Glut, 5

The synthesis of comonomer bis(ε-Lys-OBn)Glut 5 was started from Fmoc-Lys(Boc)-OH. Fmoc-Lys(Boc)-OH was first esterified with benzyl alcohol, followed by removing Fmoc group by 20% piperidine-DCM according to Agnihotri's report [30]. One gram of NH₂-Lys(Boc)-OBn 3 was dissolved in 50 mL of dry DCM, and then Glutaric acid (0.16 g) and EDCI (0.57 g) were added to the solution. The coupling reaction was initialized by adding 0.52 mL of triethylamine (Et₃N) to the solution. After stirring overnight at room temperature, the reaction was stopped with 50 mL of saturated NH₄Cl aqueous solution. The organic layer was separated, dried with Na₂SO₄, and concentrated under vacuum. The compound 4 was purified from the pale white residue by silica gel chromatography (CHCl₃/MeOH = 50/1, v/v). To remove the Boc groups, compound 4 (0.5 g) was dissolved in 30 mL of TFA/DCM (v/v, 1/1) and stirred at room temperature for 1 hour. The solvent was evaporated under vacuum, and ice-cold diethyl ether was added to precipitate the product. The white precipitate was collected by filtration and dried under vacuum to get the pure product 5 (~ 60%). ¹H NMR (400 MHz, D₂O) δ 7.27 (s, 5H), 5.05 (m, 2H), 4.25 (t, 1H), 2.79 (t, 2H), 1.95 (m, 2H), 1.65 (m, 3H), 1.53 (m, 2H), 1.26 (m, 2H). ESI-MS calcd. for C₃₁H₄₄N₄O₆ 568.7; found 569.3 (M + H)¹⁺.

Synthesis of Poly[bis(ε-Lys-OBn)Glut-PEG], 6a-d

Before the reaction, bis(ε-Lys-OBn)Glut 5 was dried in a vacuum desiccator for two days. Bis(ε-Lys-OBn)Glut 5 (19 mg) and NHS-PEG₃₄₀₀-NHS (100 mg) were transferred to a dry 5-mL flask, followed by adding 0.5 mL of extra dry DMF (water < 50 ppm). The suspension was stirred at room temperature for 10 min, and then anhydrous Et₃N was added into the reaction in nitrogen atmosphere. After stirring at room temperature or 50°C for 2-24 hours, the mixture was diluted with 50 mL of water and dialyzed against distilled water using a dialysis tubing with MWCO 14,000. The polymer solution was lyophilized, and pure polymer 6a-d was collected as white powder (60-80%). The molecular weight of polymer was determined on a Tosoh EcoSec HLC 8320GPC system equipped with a differential refractometer. The gel permeation column (Styragel®, 8 μm 300 ×7.5 mm, Waters Corporation) was calibrated using polystyrene standards and eluted with THF at a flow rate of 0.7 mL/min. As for 6b, Mw = 31.5 kDa, Mn = 21.9 kDa, Mw/Mn = 1.43. ¹H NMR (400 MHz, D₂O) δ 7.30 (s, 5H), 5.08 (m, 2H), 4.26 (t, 1H), 4.07 (m, 2H), 3.61 (m, PEG-CH₂), 3.43 (m, 2H), 2.95 (m, 2H), 2.16 (m, 2H), 2.15-2.18 (m, 5H), 1.35 (m, 2H).

Synthesis of Linker-Drug, Gly-Rapa and GlyGlyGly-Rapa

31-O-TMS Rapamycin 10

31-Trimethylsilyl Rapamycin was synthesized according to the procedure described by Shaw and Nan [31-32]. Briefly, Rapamycin (200 mg) and imidazole (150 mg) were dissolved in 25 mL of DCM. After cooling to 0°C, trimethylsilyl chloride (TMSCl) solution in DCM (1M, 1.3 mL) was slowly added into the reaction. The reaction was stirred for around 10 minutes at 0°C. After the reaction was complete, the mixture was directly applied on silica gel column and eluted with petroleum ether/acetone (v/v, 5/1). The purified intermediate 31,42-bis-O-TMS Rapamycin 9 (210 mg) was then dissolved in 20 mL of DCM, followed by adding 100 mg of imidazole/imidazole·HCl (mole ratio, 1/1). After stirring at room temperature for 3 hours, the reaction was stopped and 31-O-TMS Rapamycin 10 was purified by flash chromatography on silica gel (petroleum ether/acetone, v/v = 5/1). The overall yield for the two-step reaction is ~ 50%. ¹H NMR (400 MHz, CDCl₃) δ 6.41 (m, 2H), 6.17 (q, 1H), 6.05 (d, 1H), 5.59 (q, 1H), 5.33 (d, 1H), 5.22 (d, 1H), 5.05 (q, 1H), 4.73 (s, 1H), 4.07 (d, 1H), 3.85 (m, 2H), 3.72 (m, 1H), 3.66 (d, 1H), 3.41 (s, 3H), 3.38 (m, 3H), 3.27 (s, 3H), 3.14 (s, 3H), 2.93 (m, 1H), 2.68 (m, 3H), 2.31 (m, 2H), 2.11 (m, 1H), 1.98 (m, 3H), 0.60-1.80 (m, 41H), 0.04 (s, 9H). ESIMS calcd. for C₅₄H₈₇NO₁₃Si 986.4; found 1008.7 [M + Na]⁺.

Trt-Gly-Rapa 12

31-O-TMS Rapamycin 10 (100 mg) and Trt-Gly-OH (64 mg) were dissolved in 5 mL of DCM. After stirring at room temperature for 5 minutes, EDCI (40 mg) and DMAP (2 mg) were added to the reaction. The reaction mixture was continuously stirred for 2 hours at room temperature. Trt-Gly-Rapa(31-O-TMS) 11 was purified from the reaction using silica gel chromatography (petroleum ether/acetone, v/v = 5/1). The TMS group at the 31 site of 11 was selectively removed using inorganic acid. Briefly, compound 11 was dissolved in 4 mL of ice-cold THF, followed by adding 2 mL of 2N H₂SO₄ solution. The reaction was warmed to room temperature and stirred for 30 minutes. After the reaction was complete, the mixture was poured into 20 g of ice and neutralized by adding excess amount of NaHCO₃. The neutralized solution was extracted with AcOEt. The organic phase was washed with brine, dried and concentrated. The residue was applied on a silica gel column and purified using petroleum ether/acetone (v/v = 3/1) as the elution solution (yield, ~ 80%). H NMR (400 MHz, CDCl₃) δ 7.46 (d, 6H), 7.28 (m, 6H), 7.26 (t, 3H), 6.36 (m, 2H), 6.13 (q, 1H), 5.95 (d, 1H), 5.55(q, 1H), 5.42 (d, 1H), 5.29 (d, 1H), 5.15(q, 1H), 4.79 (s, 1H), 4.61 (m, 1H), 4.17 (d, 1H), 3.87 (m, 1H), 3.75 (d, 1H), 3.66 (m, 1H), 3.55 (d, 1H), 3.38 (m, 1H), 3.33 (s, 3H), 3.30 (s, 3H), 3.27 (m, 1H), 3.14 (s, 3H), 3.08 (m, 1H), 2.69 (m, 2H), 2.58 (m, 1H), 2.32 (m, 2H), 2.17 (s, 2H), 2.05 (m, 1H), 1.93 (m, 3H), 0.75-1.80 (m, 41H). ESI-MS calcd. for C₇₁H₉₈N₂O₁₃ 1213.5; found 1213.7 [M + H]⁺.

Gly-Rapa 13

Protecting group Trt was removed using 0.1 M HOBT in trifluoroethanol (TFE) as reported [33]. Briefly, compound 12 (50 mg) was dissolved in 3 mL of freshly prepared 0.1M anhydrous HOBT in TFE. The reaction was stirred at room temperature for 10 minutes. The process of the reaction was monitored by TLC. After the Trt group was completely removed, 100 mL of water was added to stop the reaction. The white suspension was then extracted with 100 mL of AcOEt. The organic phase was washed with brine, dried by Na₂SO₄ and concentrated under vacuum. Gly-Rapa 13 was purified from the residue using silica gel chromatography (CHCl₃/MeOH , v/v = 10/1) and yielded as white powder (~70%). H NMR (400 MHz, CDCl₃) δ 6.36 (m, 2H), 6.14 (q, 1H), 5.95 (d, 1H), 5.56(q, 1H), 5.43 (d, 1H), 5.28 (d, 1H), 5.16(q, 1H), 4.97 (s, 1H), 4.73 (m, 1H), 4.18 (d, 1H), 3.86 (m, 1H), 3.75 (d, 1H), 3.66 (m, 1H), 3.55 (d, 1H), 3.43 (m, 2H), 3.37 (s, 3H), 3.33 (s, 3H), 3.14 (s, 3H), 2.81 (m, 1H), 2.69 (m, 2H), 2.59 (m, 1H), 2.32 (m, 2H), 2.12 (s, 1H), 2.04 (m, 2H), 1.99 (m, 3H), 0.75-1.80 (m, 45H). ESI-MS calcd. for C₅₃H₈₂N₂O₁₄ 971.2; found 971.9 [M + H]⁺.

GlyGlyGly-Rapa 15

Compound 13 (20 mg) and Trt-GlyGly-OH (20 mg) were dissolved in 10 mL of dry DCM. After stirring at room temperature for 5 minutes, EDCI (10 mg) and Et₃N (8 μL) were added to the mixture. After two hours, the reaction was stopped by adding 10 mL of saturated NH₄Cl aqueous solution. The organic phase was then separated, washed with brine, dried by Na₂SO₄ and concentrated under vacuum. The intermediate Trt-GlyGlyGly-Rapa 14 was purified on silica gel column (CHCl₃/MeOH, v/v = 50/2.5). The Trt group of 14 was deprotected using the same method as described above. The final product GlyGlyGly-Rapa 15 was purified by silica gel chromatography (CHCl₃/MeOH, v/v = 5/1, then 2/1,). The yield for the two steps was 50~ 60%. ¹H NMR (400 MHz, CDCl₃) δ 6.35 (m, 2H), 6.13 (q, 1H), 6.00 (d, 1H), 5.51(q, 1H), 5.41 (d, 1H), 5.27 (d, 1H), 5.14(q, 1H), 4.66 (m, 2H), 4.23 (d, 1H), 4.01 (m, 4H), 3.89 (m, 2H), 3.65 (m, 2H), 3.57 (d, 1H), 3.31-3.35 (m, 4H), 3.14 (s, 3H), 2.96 (s, 3H), 2.89 (s, 3H), 2.62 (m, 1H), 2.31 (m, 2H), 0.75-2.00 (m, 53H). ESI-MS calcd. for C₅₇H₈₈N₄O₁₆ 1085.3; found 1085.8 [M + H]⁺.

Synthesis of rapamycin-polymer conjugate

Synthesis of Poly[bis(ε-Lys)Glut–PEG] 7

Polymer 7 was synthesized by removing the benzyl groups from carboxylic acids of polymer 6b via hydrogenation. Briefly, polymer 6b (150 mg) was dissolved in 50 mL of methanol in nitrogen atmosphere. Twenty milligrams of Pd/C (palladium loading 10%) was transferred into the reaction flask under nitrogen atmosphere. After all the Pd/C was merged into methanol, nitrogen was changed into hydrogen, and the reaction was stirred at room temperature overnight. The reaction mixture was then filtered to remove Pd/C. The filtrate was concentrated and dried under vacuum overnight to get polymer 7 in quantitative yield. ¹H NMR (400 MHz, D₂O) δ 4.20 (m, 1H), 4.10 (m, 2H), 3.61 (m, PEG-CH₂), 3.43 (m, 2H), 3.01 (t, 2H), 2.21 (t, 2H), 1.82 (m, 1H), 1.69 (m, 1H), 1.55 (m, 2H), 1.41 9m, 2H), 1.25 (m, 2H).

Synthesis of Rapamycin-polymer conjugate 8a-c

Conjugate 8a-c was synthesized by coupling linker-drugs (rapamycin, Gly-Rapa and GlyGlyGly-Rapa) with polymer 7 using EDCI/Et₃N/NHS. Briefly, polymer 7 (5 mg, 0.0015 mmol of repeat unit) and linker-drug (0.006 mmol) were dissolved in 0.5 mL of dry DMF. EDCI (10 mg), DMAP (2 mg) and NHS (1 mg) were added into the reaction and stirred for 24 hours. The polymer was then precipitated with diethyl ether (20 mL). After centrifugation, ether was poured out and the precipitate was dissolved in 5 mL of water. The insoluble solid was removed by filtering through 0.2 μm filters. The solution was dialyzed against distilled water by 14 kDa MWCO membrane at room temperature for 48 hours. Dialysis water was changed every 8 hours. The solution was free-dried to yield as a white solid (yield, 41-88%).

Synthesis of Rapamycin-polymer conjugate 8d

Conjugate 8d was synthesized using the same method as described above but the coupling reagent was changed to HATU/DIPEA. Briefly, polymer 7 (5 mg) and GlyGlyGly-Rapa 15 (6 mg) were added into 0.5 mL of dry DMF, followed by HATU (20 mg) and DIPEA (16 μL). The reaction was continuously stirred at room temperature for 24 hours. Twenty milliliter of diethyl ether was poured into the reaction to precipitate the product. The precipitate was collected by centrifugation (4,000 g, 5 min) and re-dissolved in 5 mL of water. After dialysis and free drying, the pure conjugate 8d yielded as a light brown powder (yield ~ 50%).

Determination of rapamycin loading efficacy (wt%)

The percentage of rapamycin conjugated to polymers (wt%) was determined by Ultraviolet-Visible (UV) spectrophotometry. The standard curve was generated with a series of rapamycin solution in water (λ_max = 280 nm). The polymer-drug conjugates were diluted in water, and the UV absorbance at 280 nm was measured. Rapamycin concentration was calculated from the standard curve. The percentage of rapamycin conjugated to polymer (wt%) was calculated by dividing the amount of rapamycin by the total weight of the polymer-drug conjugate.

Rapamycin release from the polymer conjugates

Aliquots of GlyGlyGly-Rapa and polymer-rapamycin conjugate 8c stock solution (10 mM in DMSO) were diluted to 200 μM in water. The solutions were immediately mixed with 100% human/rat/mouse sera, which gave a final concentration of 100 μM rapamycin in 50% sera. Forty microliters of the solution were divided into 0.2 mL Eppendorf tubes and incubated at 37 °C. At selected time intervals, serum proteins were precipitated with 120 μL of acetonitrile plus 0.1% TFA. After centrifugingation at 12,000 g for 10 min, 100 μL of the supernatant was collected and analyzed with HPLC for quantification of the released rapamycin.

The HPLC system was equipped with a Shimadzu LC-20AT pump, a SIL-10AF auto-sampler and a SPD-10A UV detector. The separation was achieved on a Waters C18 column (250 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 mL/min. The mobile phases were consisted of phase A (water, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), with a gradual increase of phase B from 50% to 90% over 20 min. The absorbance of rapamcyin was monitored at 280 nm.

In vitro cytotoxicity

In vitro cytotoxcity of rapamycin, the polymer and polymer-drug conjugates was determined according to the method we reported before [34]. Briefly, cells were seeded into 96-well plates at a density of 5,000 cells / well. Twenty-four hours after incubation, a series of drug dilutions ranging from 0.001-1000 nM were added to the wells and cultured for another 72 hours. The relative cell numbers were measured using MTT assay. To enhance the assay sensitivity, twenty-five microliters of Sorensen's glycine buffer (0.1M glycine, 0.1M NaCl, adjust pH to 10.5 with 0.1N NaOH) was added into wells according to a previous report [35]. The absorbance was measured using a DTX 880 Multimode Detector (Beckman Coulter,Inc., Fullerton, CA) at the wavelength of 570 nm. IC₅₀ was calculated by fitting a concentration – absorbance curve using Graphpad Prism 5 (Graphpad software Inc, La Jolla, CA).

Cellular uptake analysis by confocal laser scanning microscopy

Cellular uptake of the FITC-labeled polymer was performed and analyzed by confocal laser scanning microscopy as reported [36]. Briefly, PC-3 cells were seeded into 8-well Lab-Tek^TM chamber slides (Nunc Inc. Rochester, NY) and cultured for 24 hours. The culture medium was discarded, and monolayers were washed once with RPMI-1640 medium. Polymer-FITC was diluted to 10 μM in RPMI-1640 and added into the chambers. After incubation for 24 hours, the cells were washed six times with DPBS and fixed in 4% buffered formalin at room temperature for 10 min. The cells were washed three times to remove remaining formalin, and the nucleus was stained with TO-PRO-3 (Excitation/Emission: 642/661 nm) according to manufacturer's protocol (Invitrogen, Grand Island, NY). After washing, cell monolayers were mounted onto glass microscope slides using fluorescence mounting medium (Vector Lab, Burlingame, CA). The cellular uptake and distribution was analyzed using Nikon C1 laser scanning confocal microscope (Nikon Instrument Inc., Melville, NY).

Aqueous solubility assay

The aqueous solubilities of rapamycin and its conjugates were assayed by the thermodynamic method as reported before [34]. Briefly, excess amount of rapamycin or its conjugates were suspended in 100 μL of distilled water. The suspension was shaken at room temperature for 24 hours. The suspension was then centrifuged at 12,000 g for 15 min to remove undissolved drug. The supernatant was collected, and the concentration was quantified by UV spectrometry at 280 nm.

Statistical Analysis

Data were expressed as the mean ± standard deviation (SD). Difference between any two groups was determined by ANOVA. P<0.05 was considered statistically significant.

RESULTS

Novel engineering of rapamycin-polymer conjugate

In prior studies, rapamycin was specifically conjugated with small molecular-weight moieties to increase its hydrophilicity and specificity (CCI-779, APA23573 and RAD001, Figure 1A). Although the oral bioavailability was enhanced, the solubility and biodistribution profile were only slightly improved. In this paper, a rapamycin-polymer conjugate was engineered by conjugating rapamycin with a novel, PEG based, multiblock copolymer. The architecture of the polymer is composed of alternating PEG and bis(ε-Lys)Glut units. Each bis(ε-Lys)Glut unit is grafted by one or two rapamycin (Rapa) molecules and separated from other bis(ε-Lys)Glut unit by PEG (Figure 1B and C). Therefore, drugs are evenly distributed along the polymer chain and all the drugs are well separated from each other by PEG. Compared to the drug position patterns in other polymer-drug conjugates (polyaspartice acid and polymerglutamic acid - drug conjugate), this unique architecture can dramatically reduce the heterogeneity and avoid aggregation [37]. Moreover, this novel alternating, multiblock copolymer contains two drug conjugation sites in each block, which enables high drug loading capacity (wt%) (32.1% for rapamycin). Rapamycin is attached onto the polymer by tri-peptide GlyGlyGly linker which can dramatically increase drug loading efficiency. Rapamycin is linked with GlyGlyGly via a potentially cleavable ester linkage, which can be gradually released by enzymatic/chemical hydrolysis (Figure 1C).

Synthesis of multiblock copolymers Poly[bis(ε-Lys-OBn)Glut-PEG], 6a-d, and Poly[bis(ε-Lys)Glut-PEG], 7

The multiblock copolymer Poly[bis(ε-Lys-OBn)Glut-PEG] was synthesized by condensation of monomer NHS-PEG₃₄₀₀-NHS with comonomer bis(ε-Lys-OBn)Glut 5 in the presence of organic base. Bis(ε-Lys-OBn)Glut 5 is composed of one glutaric acid and two L-lysine linked at α-amino groups (Figure 2). The ε-amino group of L-Lysine was less sterically hindered and more nucleophilic than the α-amino group, therefore making it more reactive to monomer NHS-PEG₃₄₀₀-NHS. Comonomer bis(ε-Lys-OBn)Glut 5 was synthesized according to the scheme in Figure 2. Compound 3 was synthesized from 1 according to Agnihotri's report [30]. Glutaric acid was coupled with at least 2 equivalents of compound 3 using EDCI/DMAP. Compound 4 was carefully purified by silica gel flash chromatography and treated with TFA/DCM to get comonomer bis(ε-Lys-OBn)Glut 5. Polymerization was carried out in anhydrous DMF in nitrogen atmosphere. After the polymerization was initialized by organic base Et₃N, the viscosity of the solution changed dramatically in less than 1 hour. The reaction mixture became very viscous after 24 hours, which indicated successful elongation of the polymer chain. As shown in Table 1, polymerization at 50 ^oC can produce higher Mw than that at 25 °C. It might be because that high temperature can reduce the viscosity of the polymerization solution and therefore increase the condensation efficiency [38]. Polymer Mw reduced to around 20 kDa when inorganic base NaHCO₃ was used. Although both Et₃N and NaHCO₃ can deprotonate the ε-amino groups of bis(ε-Lys-OBn)Glut 5, NaHCO₃ was partially insoluble in DMF, making it less efficient in polymerization. Polymer 6a-d are highly soluble in water, THF and chloroform, but insoluble in diethyl ether and petroleum ether.

Figure 2.

Synthesis of Poly[bis(ε-Lys-OBn)Glut-PEG]. Reagents and conditions: (a). Benzyl alcohol, EDCI, DMAP; (b). 20% piperidine in DCM; (c). Glutaric acid, EDCI, DMAP; (d). TFA/DCM, rt; (e). Et₃N, DMF.

Table 1.

Polymerization of the monomer NHS-PEG₃₄₀₀-NHS with comonomer bis(ε-Lys-OBn)Glut in different conditions

Polymer	Base	Temperature	Reaction Time	Mw (kDa)	Mn (kDa)	Mw/Mn	Yield (%)
6a	Et3N	50°C	2h	20.9	15.2	1.37	71
6b	Et3N	50°C	24h	31.5	21.9	1.43	59
6c	Et3N	25°C	24h	22.8	16.1	1.41	66
6d	NaHCO3	50°C	24h	19.9	14.1	1.42	79

To graft rapamycin onto polymer, polymer 6b was deprotected by hydrogenation to liberate the carboxyl groups (Figure 3). Polymer 7 is a novel, linear multiblock copolymer containing two carboxyl groups for drug conjugation in each block. This polymer is also soluble in water, DMF, and methanol, slightly soluble in CHCl₃, but insoluble in THF and diethyl ether.

Figure 3.

Synthesis of the rapamycin-polymer conjugate. Reagents and conditions: (a). H₂, Pd/C in methanol; (b). EDCI, Et₃N, NHS.

Synthesis of the rapamycin-polymer conjugate

Rapamycin was first conjugated with polymer 7 by forming an ester bond between its hydroxyl groups and polymer's carboxyl groups in the presence of EDCI, Et₃N and NHS. Conjugate 8a was purified by dialysis with a yield around 67%. However, conjugate 8a had very low drug loading capacity (wt% ~ 0.48%). If all the carboxyl groups were conjugated with rapamycin, the theoretical maximal drug loading capacity (wt%) could be as high as 34.1%. Therefore, the hydroxyl group of rapamycin only reacted with around 1.4 percent of all the carboxyl groups in polymer 7. The low coupling efficiency could be explained partially by the steric hindrance between the polymer and rapamycin. To reduce the steric hindrance, two linkers, Gly and GlyGlyGly, were utilized to link polymer 7 and rapamycin.

Linker-drugs were synthesized according to the scheme in Figure 4. In order to selectively conjugate the linkers at 42-OH, 31-OH of rapamycin was selectively protected with TMS group according to the procedure described by Shaw and Nan [31-32]. Trt-Gly-OH was then conjugated with 42-OH using a conventional coupling method. After removing TMS at 31-OH in diluted inorganic acid H₂SO₄, Trt-Gly-Rapa was deprotected with 0.1M HOBT in TFE to get Gly-Rapa 13. Routine Trt deprotection methods, such as 1% TFA in DCM, were inapplicable because rapamycin is very sensitive to TFA. Gly-Rapa 13 was further coupled with Trt-Gly-Gly-OH using EDCI/Et₃N to give compound 14. GlyGlyGly-Rapa 15 was finally obtained by removing the Trt group in compound 14.

Figure 4.

Synthesis of GlyGlyGly-Rapa. Reagents and conditions: (a). TMSCl, imidazole, DCM; (b). imidazole/imidazole·HCl, DCM; (c). Trt-Gly-OH, EDCI, DMAP, DCM; (d). THF/2N H₂SO₄ (v/v=2/1); (e). 0.1M HOBT in TFE; (f). Trt-Gly-Gly-OH, EDCI, Et₃N, DCM.

Linker-drugs Gly-Rapa 13 and GlyGlyGly-Rapa 15 were conjugated with polymer 7 using the same coupling method as the synthesis of 8a. Conjugate 8b was obtained with a yield of 65-88%, and the weight percentage (wt%) of rapamycin in the polymer-drug conjugate was 19.5%. The highest weight percentage of rapamycin in polymer-drug conjugates was observed in conjugate 8c (wt% ~ 27.3% ) where around 80% of carboxyl groups in polymer 7 were conjugated with rapamycin. There was no significant difference in drug loading capacity when other coupling methods, such as HATU/DIPEA, are used (conjugate 8d, Table 2). Therefore, the conjugation efficiency is mainly determined by linkers rather than coupling methods.

Table 2.

Properties of the rapamycin-polymer conjugates

Polymer-drug conjugate	Conjugation reagents	Linker-Drug	Theoretical maximal drug conjugated (wt%)	Drug conjugated (wt%)	Yield (%)
8a	EDCI/Et3N/NHS	Rapamycin	34.1	0.68±0.18	57~71
8b	EDCI/Et3N/NHS	Gly-Rapa	33.4	19.5±1.8	65~88
8c	EDCI/Et3N/NHS	GlyGlyGly-Rapa	32.1	27.3±5.1	41~63
8d	HATU/DIPEA	GlyGlyGly-Rapa	32.1	24.9±3.1	50~64

Characterizations of rapamycin-polymer conjugate

The UV spectrum of conjugate 8c was scanned over the range of 190 to 400 nm and compared with free rapamycin. As shown in Figure 5, The UV spectrum of rapamycin exhibits a maximum absorbance peak at 280nm and two small peaks on either side. Similar peaks were also observed in the UV spectrum of purified conjugate 8c, which indicates that rapamycin is conjugated to the polymer chains. Beside three peaks around 280 nm, conjugate 8c also shows strong absorbance at near ultraviolet region (200 - 230 nm), which is contributed mainly by polymer 7 and minimally by rapamycin (Figure 5). The carboxyl groups in polymer 7 and double bonds in rapamycin are believed to be responsible for the UV absorbance at 200 ~ 230 nm.

Figure 5.

UV spectra of rapamycin, rapamycin-polymer conjugate 8c and polymer 7.

Proton NMR (¹H NMR) spectroscopy was also determined to prove the presence of rapamycin in the rapamycin-polymer conjugate 8c. Figure 6A shows the ¹H NMR spectrum of rapamycin. The seven groups of peaks (a - g) at low field corresponds very well to the protons of double bonds (a - e) and carbonyl groups (f, g) in rapamycin. The single peaks (h - j), between 3.0 to 3.5 ppm, corresponds to the presence of three CH₃O- groups in rapamycin. The ¹H NMR spectrum of rapamycin-polymer conjugate 8c (Figure 6C) contains peaks representing both rapamycin and the polymer chain. Peaks a - j in Figure 6C show the same chemical shifts (ppm) and splitting patterns as that in Figure 6A, indicating the presence of rapamycin in the conjugate 8c. While the polymer chain in the conjugate 8c corresponds to the peaks a’ - c’, in which peaks a’ - b’ represent bis(ε-Lys)Glut and peak c’ represents PEG₃₄₀₀ (Figure 6B).

Figure 6.

¹H NMR spectra of rapamycin (A), polymer 7 (B) and rapamycin-polymer conjugate 8c (BC) in CDCl₃ solvent.

Release of rapamycin from rapamycin conjugates

The release of rapamycin from two conjugates GlyGlyGly-Rapa and conjugate 8c was evaluated in mouse, rat and mouse serum. Rapamycin conjugates were incubated with 50% of different serum at 37 °C for 4 hours. The release profile of rapamycin was monitored using HPLC. In mouse serum, the half-life of releasing rapamycin from GlyGlyGly-Rapa is approximately 0.5 hours. In contrast, the half-life increases to 4 hours in rat serum. A minimal release was observed in human serum where only 10% of rapamycin was released after 4 hours (Figure 7). The species-dependent release profile is due to the metabolic differences between species [39]. The similar phenomenon was also observed in Wortmannin-Rapamycin conjugate [27]. The release of rapamycin from polymer-rapamycin conjugate 8c is much slower than that of GlyGlyGly-Rapa. After 4-hour incubation, only ~20%, ~9% and ~3% of rapamycin were released from conjugate 8c in mouse, rat and human serum, respectively. The bulky polymer structure in polymer-rapamycin conjugate dramatically increased the steric hindrance of the ester linker between rapamycin 42-OH and GlyGlyGly, which makes ester bond less accessible by esterase and therefore decreases the hydrolysis rate [40]. In addition, the release of rapamycin from polymer-rapamycin reached its plateau at 1 hour and 2 hours in mouse and rat serum, respectively. These premature plateau-release profiles can be attributed to the instability of rapamycin in sera. The half-life of rapamycin in rat plasma has been reported to be only 2.2 hours, and a shorter half-life would be expected in mouse plasma due to its higher metabolic rate [39, 41]. Therefore, the plateau was reached when the degradation rate of rapamycin was equal to its release rate.

Figure 7.

The release profiles of rapamycin from GlyGlyGly-Rapa (A) and conjugate 8c (B) in different sera.

In vitro activity of rapamycin and its conjugates

The in vitro cytotoxicity of polymer 7, rapamycin, GlyGlyGly-Rapa and conjugate 8c was determined in prostate, breast, and cervical cancer cell lines using a MTT assay. No obvious cytotoxicity (IC₅₀ > 100 μM) was observed in polymer 7 treated cells, indicating that polymer 7 is not toxic to these cancer cell lines. Rapamycin showed potent in vitro cytotoxicity to all cancer cell lines (Table 3). The IC₅₀ values of rapamycin in all seven cancer cell lines ranged from 0.1 to 5.2 nM. Compared to breast cancer cell lines, prostate and cervical cancer cell lines were much more sensitive to rapamycin treatment. GlyGlyGly-Rapa was 2- to 36-fold less potent than rapamycin with the exception of MCF-7/HER2, in which similar potency was observed. Given the fact that GlyGlyGly-Rapa is an ester prodrug of rapamycin, and hydrolysis is required for its activity, it is reasonable that GlyGlyGly-Rapa shows lower potency than rapamycin. The IC₅₀ values of conjugate 8c are in a range of 25 to 50 nM. Although it is less potent than free rapamycin, conjugate 8c still exhibits low nanomolar IC₅₀ values against all cancer cell lines tested.

Table 3.

In vitro cytotoxicity (IC₅₀, nM). The cancer cells were treated with the compounds for 72 hours before MTT assay. Values shown are mean ± SD.

Cancer type	Cell line	Polymer 7a	Rapamycin	GlyGlyGly-Rapa	Conjugate 8cb
Prostate	LNCaP	>100,000	0.9±0.9	4.1±0.3	51.2±0.6
	PC-3	>100,000	0.8±0.7	2.3±0.6	29.9±0.7
	DU145	>100,000	0.1±0.5	5.6±0.5	30.6±0.6
Breast	SK-BR-3	>100,000	4.4±0.3	9.2±0.6	40.7±0.7
	MCF-7	>100,000	5.2±0.3	10.3±0.5	26.3±0.4
	MCF-7/HER2	>100,000	3.3±0.2	3.9±0.4	25.9±0.5
Cervical	HeLa	>100,000	1.0±0.1	36.5±0.1	47.4±0.1

^aIC₅₀ is calculated based on molar concentration of repeated units.

^bIC₅₀ of polymer rapamycin conjugate 8c is expressed in terms of rapamycin equivalents.

Cellular uptake and distribution of the rapamycin-polymer conjugates

FITC ethylenediamine (FITC-NH₂) was conjugated to polymer 7 using the same coupling method (EDCI/NEt₃/NHS) as conjugate 8a-c. The weight percentage (wt%) of FITC in the labeled polymer 7 is 4.5% (Data not shown). To determine the cellular uptake and distribution, The FITC-labeled polymer was incubated with PC-3 cells in serum-free medium for 24 hours. Punctuated staining pattern was observed in PC-3 cells after 24-hour treatment (Figure 8). Other fluorescently labeled polymers, such as β-cyclodextrin polymer and PEG, were reported to share the similar cellular uptake and distribution pattern [36, 42]. Polymers are believed to enter cells via endocytosis because the punctate vesicular distribution of fluorescence indicated a lysosomal localization. The uptake of FITC-NH₂ in PC-3 cells was also examined. Because FITC-NH₂ is a membrane non-permeable fluorescent dye, no fluorescence was observed even after 24-hour incubation (data not shown) [43].

Figure 8.

Cellular uptake of FITC labeled polymer (Polymer-FITC) and FITC ethylenediamine (FITC-NH₂) by confocal microscopy. PC-3 cells were treated with Polymer-FITC and FITC-NH₂ in serum free RPMC-1640 media for 24 hours. Cell nucleus was stained with TO-PRO-3 and imaged in two fluorescent channels.

Conjugation with polymer dramatically enhanced the solubility of rapamcyin

Due to the hydrophilic PEG component, polymer 7 is highly soluble in water (Table 4). The aqueous solubility of rapamycin is only around 0.034 mg/mL at room temperature. After conjugated to polymer 7, its solubility increases to 21.061 mg/mL which is more than 600 times higher than free rapamycin. The aqueous solubility of the rapamycin-polymer conjugate 8c is around 78 mg/mL.

Table 4.

Aqueous solubility of polymer 7, Rapamycin and conjugate 8c

Compound	Aqueous solubility (mg/mL)	Aqueous solubility (Rapamycin equivalents)
		mg/mL	mM
Polymer 7	100.332±22.278	−/−	−/−
Rapamycin	0.034±0.006	0.034±0.006	0.037±0.007
Conjugate 8c	78.000±12.165	21.061±3.285	23.014±3.594

DISSCUSION

Rapamycin has been examined alone or in combination with other drugs for treatment of various cancers in clinical studies [3-5]. Although it has shown promising therapeutic effects, its clinical development was interrupted by poor aqueous solubility and preferential distribution in RBCs. Various formulations and conjugates, such as cosolvent system, polymeric micellar formulation and some rapalogues, have been developed to overcome the limitations, [8, 15, 17]. All these approaches can only partially increase the solubility, but has little effect on the blood distribution and pharmacokinetics. Therefore, the objective of this study is to develop a rapamycin-polymer conjugate that not only enhance the solubility but also improve the biodistribution profiles of rapamycin.

In this study, rapamycin was conjugated with a novel, linear, PEG containing multiblock copolymer. The polymer was synthesized via the condensation of a difunctionalized PEG monomer (NHS-PEG₃₄₀₀-NHS) with a comonomer bis(ε-Lys-OBn)Glut 5 containing two pendant carboxyl groups for drug conjugation. Comonomer bis(ε-Lys-OBn)Glut 5 is small in size and symmetric in structure, which is expected to enhance the condensation efficiency. The purity of the comonomer has a significant impact on achieving high M_W polymers. The presence of impurities can terminate the chain elongation, leading to low MW polymers [26]. After precipitation in diethyl ether twice, bis(ε-Lys-OBn)Glut was obtained with a purity of ~ 97%. Beside impurity, moisture can also dramatically decrease the M_W of the polymer. Polymerization in non-drying solvent or with non-drying comonomer would only generate short polymer chains with M_W of ~18 kDa (data not shown).

This study represents the first attempt to conjugate rapamycin with a polymer. Rapamycin is a structurally complicated natural product which contains three hydroxyl groups. Although the tertiary alcohol at position 13 is highly hindered and unreactive, rapamycin contains two secondary hydroxyl groups at position 31 and 42 which are accessible for esterification and etherification. Directly reacting with acrylating agents would produce a mixture of 31,42-bis-acrylated product and 31- or 42-monoacrylated products, which results in inevitable heterogeneity of the rapamycin-polymer conjugate. In this study, we achieved the regioselective conjugation of rapamycin at 42-OH by first protecting the 31-OH with TMS and then synthesizing 42-monoacrylated rapamycin via esterification. Another challenge of rapamycin conjugation is its instability in both acidic and basic conditions which make it intolerable to many chemical reactions. Gly-Rapa was firstly attempted to synthesize from Fmoc-Gly-Rapa, but rapamycin was quickly degraded when deprotecting Fmoc in 10% piperidine in DCM. Removing Boc from Boc-Gly-Rapa was also problematic due to its sensitivity to TFA (even 1% TFA in DCM). Gly-Rapa was finally obtained with a very low yield (~ 10%) from Trt-Gly-Rapa by deprotecting Trt in THF/2N H₂SO₄ (v/v = 2/1, overnight). However, the yield was dramatically increased to 60~80% when the deprotection method was switched to an uncommonly used, but mild condition (0.1N HOBT in TFE, 10 min).

Conjugation of rapamycin to the polymer was achieved by coupling a linker to rapamycin at position 42-OH, and subsequently grafting the linker-Rapa at the pendant carboxyl groups of this multiblock copolymer. The expected mechanism of drug release from the rapamycin-polymer conjugate is that tumor esterase would hydrolyze the 42-ester bond and release free rapamycin from the polymer backbone. Insertion of linkers, such as Gly or GlyGlyGly, between rapamycin and the polymer backbone is believed to accelerate the drug release [25-26, 37]. Moreover, these amino acids or peptide linkers can transform the hydroxyl group of rapamycin at position 42 to a more reactive amino end group, which could dramatically increase the drug loading capacity (Table 2). As shown in Table 2, directly conjugating rapamycin to the polymer can only load a small amount of drug (wt% ~ 0.68). In contrast, the drug loading capacities increased to 19.5% and 27.3%, respectively, by inserting Gly and GlyGlyGly between rapamcyin and the polymer. The use of longer linker, such as GlyGlyGly, resulted in a higher drug loading capacity because the long and flexible linker may reduce the steric hindrance between the polymer and rapamycin during coupling reaction. The resulting polymer rapamycin conjugate 8c dissolves freely in water. Its aqueous solution is stable and does not precipitate/change color when it is not exposed to strong ionic strength or light.

The most striking observation of this study is the great solubility of rapamycin after conjugating with the polymer. Rapamycin is practically insoluble in water (0.034 mg/mL), but its solubility increases to 21.061 mg/mL after conjugating with the polymer. This solubility meets the requirement for i.v. formulations in animal studies, in which the minimal dose concentration is 3 ~ 6 mg/mL, and clinical studies, in which the minimal dose concentration is 1 mg/mL [8]. The backbone of polymer 7 contains repeating units of two components, PEG₃₄₀₀ and bis(ε-Lys)Glut, in which PEG₃₄₀₀ attributes to the aqueous solubility. Moreover, PEG₃₄₀₀ is also expected to reduce the toxicity and immunogenicity of the polymer [44]. As shown in Table 3, polymer 7 does not show obvious toxicity in various cultured tumor cells.

The release of rapamycin from the polymer-drug conjugate in the presence of serum was observed (Figure 7). The polymer-rapamycin conjugate exhibits different release rates in the serum of different species, which indicates that the release of rapamycin is mainly mediated by enzymatic hydrolysis other than pure chemical hydrolysis. Bulk structure of the polymer dramatically hinders the enzymatic hydrolysis and therefore slowdowns the rapamycin release in the serum, which can avoid the redistribution of rapamycin into RBCs. Same to other polymer-drug conjugates, the rapamycin-polymer conjugate is taken up by tumor cells via the endocytotic pathway (Figure 8) [36, 42]. In the intracellular compartment endosomes and lysosomes, rapamycin-polymer would be attached by peptidase/esterase and release the drug rapamycin.

In conclusion, a novel, linear, PEG-based multiblock copolymer was synthesized and utilized as a carrier for hydrophobic drug rapamycin. The rapamycin-polymer conjugate shows significantly increased solubility in water and potent cytotoxicity against a panel of cancer cell lines. It also demonstrates that the polymer-drug conjugate can be taken up by tumor cells. The result of the uptake study indicates that the lysosome is the main site of the intracellular localization of the polymer-drug conjugate. Thus, in this study we reported a novel rapamycin conjugate which holds great promising for treatment of a wide variety of tumors. Further studies of this rapamycin conjugate are ongoing in several animal xenograph models to explore its efficacy in vivo.

Abbreviations

FKBP12

FK-binding protein 12

mTOR

the mammalian target of rapamycin

PEG

poly(ethylene glycol)

RBCs

red blood cells

Rapalogues

rapamycin analogues