Programmed Hydrolysis in Designing Paclitaxel Prodrug for Nanocarrier Assembly

Nanocarriers delivering prodrugs are a way of improving in vivo effectiveness and efficiency. For therapeutic efficacy, the prodrug must hydrolyze to its parent drug after administration. Based on the fact that the hydrolysis is impeded by steric hindrance and improved by sufficient polarity, in this study, we proposed the PTX-S-S-VE, the conjugation of paclitaxel (PTX) to vitamin E (VE) through a disulfide bridge. This conjugate possessed the following advantages: first, it can be encapsulated in the VE/VE2-PEG2000/water nanoemulsions because of favorable hydrophobic interactions; second, the nanoemulsions had a long blood circulation time; finally, the concentrated glutathione in the tumor microenvironment could cleave the disulfide bond to weaken the steric hindrance and increase the polarity, promoting the hydrolysis to PTX and increasing the anticancer activity. It was demonstrated in vitro that the hydrolysis of PTX-S-S-VE was enhanced and the cytotoxicity was increased. In addition, PTX-S-S-VE had greater anticancer activity against the KB-3-1 cell line tumor xenograft and the tumor size was smaller after the 4th injection. The present result suggests a new way, use of reduction, to improve the in vivo anticancer activity of a prodrug for nanocarrier delivery by unshielding the ester bond and taking off the steric block.

the poor hydrolysis of PTX-VE might be that the hydrolytic site (C-2' hydroxyl group in PTX) is sterically shielded by the VE. To test this hypothesis, PTX-SEE (the VE group substituted by a methyl group) was synthesized. Both PTX-VE and PTX-SEE are hydrophobic. As shown in Fig. 3a, approximately 20.7% of PTX-SEE was hydrolyzed to free PTX by 48 h, a much higher percentage than for PTX-VE. This indicates that steric hindrance could be a major reason for impeded hydrolysis.
In addition to this, Markovic et al. established a good correlation (R = 0.9924) between solvolytic rate constants and polarity, indicating enhanced polarity could improve hydration for a prodrug 32 . It  The prodrug will experience a two stepped hydrolysis to overcome the steric hindrance and polarity hurdles. The disulfide bond will be cleaved in tumor cells because the concentration of GSH in tumor cells is much higher than in blood plasma. This initial hydrolysate will then be further hydrolyzed to PTX because of the increased polarity.
is reasonable to assume that the hydrolysis rate is dependent on polarity to some extent. Therefore, we conjugated PTX and succinic acid directly to produce PTX-SA. In comparison with PTX-SEE, PTX-SA had increased polarity, which was evidenced by the retention time (6.7 min for PTX-SA and 31.7 min for PTX-SEE) in a reversed phase chromatography system ( Figure S6). As expected, PTX-SA had a higher hydrolytic profile than PTX-SEE, suggesting that sufficient polarity might be the other reason for hydrolysis.
Therefore, decreased steric hindrance and enhanced polarity favor increased hydrolysis.
Cytotoxicity demonstration. For PTX, the hydroxyl group at the C-2' position is important for its anticancer activity and it must therefore be exposed to the external environment to have its effect 16,17 . Thus, the hydrolytic rate for a PTX prodrug may therefore influence its in vitro cytotoxicity. In this research, the cytotoxicity was investigated on the KB-3-1 cell line using the MTS method. Free PTX, PTX-SA, PTX-SEE, and PTX-VE were added to the cells, respectively, with concentrations ranging from 0.1-0.5 μ M. As shown in Fig. 3b On the basis of the analysis above and considering the redox nature of the tumor microenvironment 33 , we undertook to synthesize PTX-S-S-VE (PTX conjugated with VE via a disulfide bond) because the sufficient GSH 34,35 in tumor cells will cleave the bridge and promote the hydrolysis to free PTX. As shown in Fig. 4, the hydrolysis rate was accelerated with GSH concentration increased. In addition, compared with PTX-VE, the in vitro hydrolysis was improved and its anticancer activity was increased.

Preparation, characterization and storage stability of the NES. PTX-VE/VE/VE 2 -PEG 2000
NES has been prepared because of favorable affinity for PTX-VE, VE, and VE 2 -PEG 2000 . In this study, PTX-S-S-VE/VE/VE 2 -PEG 2000 NES was also prepared successfully. The particle size, zeta potential, and morphology were characterized. As shown in Fig. 5, for both NES, the particles were of similar spherical shape and size (100-150 nm). Consistent with the TEM images, the dynamic light scattering results showed that both NES were about 130 nm (Table 1, and Figure S8-9). The small PDI indicated that the particles were of a narrow size range. It was demonstrated that the PTX-S-S-VE/VE/VE 2 -PEG 2000 NES was physically stable for at least 12 months (Table S1). Therefore, the PTX-S-S-VE/VE/VE 2 -PEG 2000 NES was suitable for in vivo studies.
Tissue distribution. To evaluate the in vivo performance, the CD1 mice, in the biodistribution study, were administrated intravenously a single dose equivalent of 20 mg/kg PTX. The mice were sacrificed at 4 h and the tissues were harvested. The biodistribution of free PTX and PTX prodrugs are shown in Fig. 6a,b, respectively. For PTX-VE NES, free PTX was only found in the liver, with only a small amount being found in other tissues. This was because of the low cleavage rate of the ester bond, and indicated similar in vivo hydrolytic behavior to that seen in vitro. On the other hand, PTX-VE NES showed high prodrug accumulation, including higher than that of PTX-S-S-VE NES, with liver (3794.0 ± 1241.6 ng/g) being the major distribution tissue followed by spleen (1753.1 ± 666.7 ng/g) and then kidney (1351.4 ± 179.3 ng/g). The reason for this could be attributed to matching properties of VE oil and the VE group in PTX-VE, as well as PTX-VE being less susceptible to hydrolysis than PTX-S-S-VE. Importantly, it exhibited comparable prodrug distribution in tumors for PTX-VE NES and

Table 1. Particle sizes and zeta-potentials of PTX-VE/VE and PTX-S-S-VE/VE NES (data are expressed as the mean ± SD, n = 3 for each measurement).
PTX-S-S-VE NES. In the case of PTX-S-S-VE NES, greater free PTX accumulation was seen than for Taxol ® in most tissues. The distribution was statistically higher than Taxol ® in plasma (p < 0.05), liver (p < 0.05), spleen (p < 0.05), kidney (p < 0.05) and tumor (p < 0.05, Student's t-test, paired, two sided). This could be because PTX-S-S-VE and VE are molecularly matched resulting in longer circulation of the PTX-S-S-VE NES in the blood and hence longer in vivo retention time than for the Taxol ® . In addition, the GSH in blood, despite being of much lower concentration than in the tumor, could start the hydrolytic program to some extent, leading to enhanced hydrolysis. The in vivo distribution results indirectly reflect the hydrolytic properties of the prodrugs.

Tumor growth inhibition.
To evaluate the in vivo therapeutic effect of the formulations, tumor growth inhibition was studied in a KB-3-1 cells subcutaneous model in Balb/C nude mice. As shown in Fig. 7, the control group showed very rapid tumor growth. There was no significant reduction of the tumor volume in mice treated with PTX-VE/VE/VE 2 -PEG 2000 NES (p > 0.05, Student's t-test, paired, two sided). However, the mean tumor volume was slightly smaller than for the controls. Not surprisingly, the greatest antitumor activity was observed when the mice were treated with PTX-S-S-VE/VE/VE 2 -PEG 2000 NES. For this formulation, the tumors were diminished before the fifth injection. Similarly, Taxol ® also showed a strong tumor inhibition effect and only one mouse was found to still bear the tumor by the 15 th day. In addition, no weight loss occurred in all groups (Fig. S10).

In vivo toxicity
Histopathological examination. In order to determine if the accumulation of the formulations is able to damage normal tissues, the histopathological of the heart, liver, spleen, lung, and kidney for each treatment group was examined. The results were shown in Fig. S11. Lesions were not found in the PTX-VE/ VE and PTX-S-S-VE/VE NES treated group. However, it is clear that the kidney, in Taxol ® treated group, suffers from dilatation and congestion for renal interstitial vascular.       under nitrogen atmosphere for 7 h. The resulting mixture was filtered to remove N, N-dicyclohexylurea (DCU) and the filtrate was dried under vacuum. The residue was purified using silica gel column chromatography, eluting with a chloroform-methanol solution with gradually increasing methanol content. The eluting solvent was removed under vacuum to give 95.9 mg of PTX-VE conjugate with the total yield of 60%. The purity was 99.2%, analyzed by the HPLC method. Compound concentration was determined at a ultraviolet wavelength of 228 nm and the injection volume was 20 μ L. Gradient elution was applied with a mobile phase of acetonitrile (ACN) and water. The initial mobile phase composition was 40% ACN from 0-36 min, and a linear gradient was applied to reach a composition of 100% ACN after a further 1 min. This was maintained for 30 min and then returned to initial conditions within a further 1 min and equilibrated for 9 min. Total run time was 77 min.

Synthesis of PTX
To analyze the hydrolysis of the prodrugs (PTX-VE and PTX-S-S-VE), the initial mobile phase composition (40% ACN) was maintained from 0-12 min, then a linear gradient was applied to reach a composition of 100% ACN after a further 1 min. This was maintained for 23 min and then returned to initial conditions within a further 1 min. VE (20 μ M), TPGS (1.8 μ M) and VE 2 -TPGS (0.34 μ M) were added to an Eppendorf Safe Lock Tube TM (1.5 mL) (Next Advance Inc., Averill Park, NY) and the chloroform removed under nitrogen flow and further by vacuum pumping. Water (600 μ L) was then added. The mixture was probe sonicated for about 1 min, and the initial NES were mixed with 0.5 g of beads (one third × 1.0 mm in diameter and two thirds × 0.5 mm in diameter). The NES were placed in a BBY24M Bullet Blender Storm (Next Advance Inc., Averill Park, NY) for homogenization.

Hydrolysis.
The particle size and zeta potential of the NES were determined using a Malvern ZetaSizer ® Nano ZS (Westborough, MA) three times. In addition, the morphology was observed with a JEOL 100CX II TEM (Tokyo, Japan).
The PTX-S-S-VE/VE/VE2-PEG2000/water NES was stored at 25 °C for 12 months. At predetermined time intervals, samples were withdrawn and evaluated, including the physical appearance, particle size, polydispersity index (PDI), and encapsulation efficiency (EE). These studies were performed in triplicate and data were expressed as mean ± SD.
Animal studies. All animal research work was approved by the University of North Carolina at Chapel Hill's Institutional Animal Care and Use Committee and were performed in accordance with relevant guidelines and regulation.
Biodistribution. Balb/C nude mice (6-8 weeks) were used to evaluate the in vivo biodistribution of the formulations. The xenograft models were established by subcutaneous injection of KB-3-1 (5 × 10 6 ) cells into the right flanks of the mice. Fifteen mice were randomly divided into three groups (n = 5 for each group) to receive Taxol ® , PTX-VE/VE/VE 2 -PEG 2000 /water or PTX-S-S-VE/VE/VE 2 -PEG 2000 /water NES, respectively. The formulations were administered intravenously via the tail vein at a single dose of 20 mg/kg PTX equivalent. After 4 h, the mice were sacrificed, and the plasma, heart, liver, spleen, lungs, kidney, and tumor were collected. The tissues were homogenized with 300 μ L of HPLC initial mobile phase (ACN:water = 40:60, w/w) using the BBY24M Bullet Blender Storm. The supernatant was obtained by centrifuging the tubes. The PTX and/or prodrugs (PTX-VE and PTX-S-S-VE) were extracted using methyl tert-butyl ether, with PTX-SEE (11.7 μ M) as internal standard. The organic phase was transferred to a glass tube and evaporated under nitrogen flow. The residues were redispersed using the initial mobile phase 40:60 (ACN:water, v/v). Analysis was carried out by the HPLC method described earlier.
In vivo anticancer effect. The KB-3-1 tumor bearing nude mice were randomly divided into four groups (n = 5 for each group) to intravenously receive PTX equivalent doses (5 mg/kg) of Taxol ® , PTX-VE NES, PTX-S-S-VE NES or physiological saline. The treatments were started on the third day after inoculation, and tumor sizes and body weights were monitored every second day. Tumor volume was calculated as V = 0.5× (L × W 2 ), where V represents tumor volume, L the larger perpendicular diameter and W the smaller one.
Histopathological examination. The histopathological examinations were conducted on the organs of the sacrificed mice. The tissues, including heart, liver, spleen, lung, and kidney, were collected, rinsed with normal saline, fixed in 10% formalin, and then stained with hematoxylin and eosin. Finally, the sections were observed under a microscope for histopathological evaluations.
Measurement of serum level. Ten CD1 mice were used for the analysis of serum biochemistry. The mice were randomly divided into two groups (n = 5 for each group) to receive Taxol ® and PTX-S-S-VE/ VE/VE 2 -PEG 2000 /water NES, respectively. The blood samples were collected by enucleating the eyeball after mice received 5 times 10 mg/kg PTX equivalent every second day, and the serum was obtained by coagulating and then centrifuging. Subsequently, the serum samples were analyzed using an AU640 blood biochemical analyzer (Olympus Co., Tokyo, Japan). The serum biochemistry analysis involved the determination of aspartate aminotransferase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN).

Hemolysis.
To test the potential toxicity of the NES, hemolysis was evaluated. Blank blood was obtained by orbital collection from the CD1 mice. Erythrocytes were immediately obtained by centrifuging the whole blood and washing twice with PBS (pH 7.4) to remove plasma and serum. The isolated erythrocytes (3 × 10 10 cells) were resuspended and incubated with the formulations at various concentrations at 37 °C for 1 h. The samples were then centrifuged for 10 min at 16000 g, and 100 μ L supernatants were diluted with an equal volume of PBS and measured for optical density (OD) at 570 nm. A complete hemolytic sample, which was used as a control, was prepared by sonicating blood for 3 min. The percentage of hemolysis was calculated as (OD test -OD formulation ) /OD control × 100% 36 .
Statistical analysis. All data were expressed as mean ± SD and were analyzed statistically using a one-way analysis of variance (ANOVA) or a two tailed Student's t-test. The differences were considered statistically significant if the p value was less than 0.05 (p < 0.05).

Conclusion
In this study, by giving a comparison of hydrolytic release and cytotoxicity for the three conjugates (PTX-VE, PTX-SA, and PTX-SEE), we have found that polarity and steric hindrance are two major factors influencing anticancer activity. Thus, we have designed a programmably hydrolyzable PTX prodrug, PTX-S-S-VE, which undergoes two subsequent hydrolysis steps and releases active drug. Like the previous PTX-VE, PTX-S-S-VE can also be encapsulated in VE/VE 2 -PEG 2000 /water NES for intravenous administration. The in vivo distribution for the two NES formulations varied a lot. In nude mice, PTX-S-S-VE/VE/VE 2 -PEG 2000 /water NES had better anticancer activity against KB-3-1 cell line tumors than PTX-VE/VE/VE 2 -PEG 2000 /water NES. Both the hemolysis activity was reduced compared with Taxol ® . In conclusion, the programmably hydrolyzable PTX prodrug that we have produced demonstrates a bright future for controlling drug release in the treatment of cancer.