Hyaluronidase-incorporated hyaluronic acid-tyramine hydrogels for the sustained release of trastuzumab Keming Xu, Fan Lee, Shujun Gao, Min-Han Tan, Motoichi Kurisawa PII: DOI: Reference:
S0168-3659(15)30061-4 doi: 10.1016/j.jconrel.2015.08.015 COREL 7795
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
10 April 2015 24 July 2015 6 August 2015
Please cite this article as: Keming Xu, Fan Lee, Shujun Gao, Min-Han Tan, Motoichi Kurisawa, Hyaluronidase-incorporated hyaluronic acid-tyramine hydrogels for the sustained release of trastuzumab, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.08.015
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ACCEPTED MANUSCRIPT Hyaluronidase-incorporated hyaluronic acid-tyramine hydrogels for the sustained release of trastuzumab
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Keming Xu, Fan Lee, Shujun Gao, Min-Han Tan, Motoichi Kurisawa*
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Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669 *Corresponding author. Tel.: +65-6824-7139; fax: +65-6478-9083 E-mail address:
[email protected] (M. Kurisawa).
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Abstract We developed an injectable hydrogel system for the sustained release of protein drugs that incorporated both protein drugs and hyaluronidase. Trastuzumab and hyaluronidase were incorporated in hydrogels composed of hyaluronic acid-tyramine (HA-Tyr) conjugates through the enzymatic crosslinking utilizing hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Through electrostatic interactions with the HA, trastuzumab was retained in the hydrogel to minimize its burst release. Hyaluronidase was incorporated in the hydrogel to release trastuzumab from the hydrogels. The hydrogels were degraded and showed sustained release of trastuzumab in phosphate buffer over four weeks in vitro. Both the rates of drug release and gel degradation were controlled by the concentration of hyaluronidase. Trastuzumab released from the hydrogels inhibited the proliferation of BT-474 cells in vitro. In an animal model, the single subcutaneous injection of a mixture solution of HA-Tyr conjugates, H2O2, HRP, trastuzumab and hyaluronidase inhibited tumor growth significantly, whereas injection of trastuzumab alone at the same dose failed to do so. Compared to trastuzumab alone, the hyaluronidase-incorporated HA-Tyr hydrogels improved the pharmacokinetic profile of trastuzumab in the plasma of mice. Furthermore, they were fully degraded over two weeks, and the formation of fibrous capsules was not observed in mice. Keywords: hydrogel, hyaluronic acid, hyaluronidase, sustained release, trastuzumab 1. Introduction Recently, injectable hydrogels have attracted much attention for the delivery of therapeutic proteins [1-3]. By preserving the native structures of proteins in a water-abundant matrix, hydrogels are ideal reservoirs for protein drugs and are expected to improve the drug release profiles. Injectable hydrogels are especially useful in clinical applications, as surgeries are not required and their administration is simple. The utilization of hydrogels with an optimized drug release profile would enhance the efficacy of the drug, reduce the frequency of drug administration, and improve the patient’s compliance. Although significant progress has been made in the field [4-7], several challenges still exist that need to be fully addressed. Firstly, as the mesh size of most hydrogels is much larger than the hydrodynamic diameters of proteins [8], it is often difficult to retain drugs in the hydrogel matrix and minimize their burst release. Secondly, since proteins are fragile and prone to denaturation, crosslinking processes need to be optimized to ensure the intactness of the protein. Finally, fibrous capsules are often formed around the hydrogels in vivo, which could hinder the release of drug, lower the degradability of hydrogel, and potentially cause chronic inflammation [9].
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The demand for a better drug delivery system has surged with the development of new drugs in the pharmaceutical industry. Monoclonal antibodies have been widely used in the treatment of cancer, yet problems such as their high costs and side effects hinder the advancement of these drugs in clinical practice. Trastuzumab, also known as Herceptin, represents one class of antibody drugs widely used for the treatment of breast cancers [10-12] which are human epidermal growth receptor 2 (HER2)-positive [13, 14]. It is administered once every three weeks over a period of one year for patients diagnosed with early-stage breast cancer [15]. The usual route of administration is intravenous (IV) infusion, which requires trained personnel and a dedicated infusion facility. It usually takes 30 to 90 minutes for one infusion, and additional time is required for post-infusion observation. Furthermore, infusion-related reactions and complications can occur in patients. Recently, subcutaneous (SC) injection has been explored as an alternative method for the administration of trastuzumab. Ismael G. et al. have reported a Phase III clinical trial that showed SC administration of trastuzumab, with recombinant human hyaluronidase (rHuPH20) [16] as excipient, offered a pharmacokinetic profile, efficacy and safety that was not inferior to IV administration [17]. Compared to IV infusion, SC treatment is less technically demanding and takes less than 5 minutes. Furthermore, 88.9% of patients preferred SC treatment in a study that examined patients’ preferences of SC administration versus conventional IV infusion, whereas only 9.6% preferred IV treatment [18]. To further enhance the efficacy of trastuzumab through the SC route, we utilized hydrogels as the delivery vehicles and exploited the enzyme-triggered trastuzumab release from the hydrogels.
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Hyaluronic acid (HA) is a naturally-occurring, biodegradable polymer with well-known biocompatibility [19, 20]. We have reported an injectable hydrogel system composed of HAtyramine (HA-Tyr) conjugates for various biomedical applications [21, 22]. The hydrogels were formed through the oxidative coupling of Tyr moieties, which were catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). In this study, we designed an injectable HATyr hydrogel system that incorporates both trastuzumab and hyaluronidase for the sustained release of trastuzumab for breast cancer treatment. Under physiological conditions, HA-Tyr is highly anionic, whereas trastuzumab with an isoelectric point of 8.45 is cationic [23]. Therefore, it is expected that the initial burst release of trastuzumab from HA-Tyr hydrogels would be minimized by immobilization of the protein in the hydrogel matrix through electrostatic interactions. Although hyaluronidase is present in the human bodies, its concentration in the plasma is only 60 ng/ml [24]. Thus we incorporated hyaluronidase into the hydrogel to control the degradation of hydrogel, which in turn would promote the release of trastuzumab. We expect that HA-Tyr hydrogels can release trastuzumab in a sustained manner through the enzymetriggered degradation of hydrogels (Fig. 1), as the hyaluronidase catalyzes the hydrolysis of α-Nacetyl-D-glucosaminidic linkages in HA. The advantages of this injectable system are: (i) minimized burst release through electrostatic interaction between HA and trastuzumab; (ii) tunable rates of drug release and gel degradation by controlling the hyaluronidase concentration; and (iii) transient presence of the hydrogel which avoids long-term discomfort and possible side effects for patients. In this study, we first examined the drug release and degradation profiles of HA-Tyr hydrogels with varying concentrations of hyaluronidase. Next, we studied the anti-proliferation effects of trastuzumab released from these hydrogels using breast cancer cell line BT-474 in vitro. In a BT-474-xenografted nude mouse model, we investigated the inhibitory effect of HA-
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Tyr hydrogels incorporating hyaluronidase in comparison to free trastuzumab on tumor growth. Histological and immunohistochemical analyses were utilized to examine the proliferation and apoptosis of tumor cells in the tissue. Pharmacokinetic studies were performed to measure the plasma concentration of trastuzumab after the treatment with hyaluronidase-incorporated hydrogels. Finally, we assessed the tissue reaction to HA-Tyr hydrogels with or without hyaluronidase.
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Fig. 1. The design of an auto-degradable HA-Tyr hydrogel that incorporates hyaluronidase for the sustained release of trastuzumab.
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2. Materials and methods 2.1. Materials Sodium hyaluronate (HA) (MW = 90 kDa, density = 1.05 g/cm3) was kindly donated by JNC Corporation (Tokyo, Japan). Hydrogen peroxide (H2O2, 30 wt.%) and hyaluronidase (439 unit/mg) from bovine testes were purchased from Sigma-Aldrich (MO, USA). Horseradish peroxidase (HRP, 100 unit/mg) was obtained from Wako Pure Chemical Industries (Osaka, Japan). RPMI-1640 medium was purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) and the alamarBlue assay kit were obtained from Life Technologies (CA, USA). Trastuzumab was purchased from Roche (Basel, Switzerland). The human IgG ELISA kit was purchased from ICL lab (OR, USA). Phosphate buffered saline (PBS, 150 mM, pH 7.3) was supplied by media preparation facility in Biopolis, Singapore. Hyaluronic acid-tyramine (HATyr) conjugates were synthesized and characterized by 1H NMR as previously described [22]. 2.2. Hydrogel preparation and rheology measurement HA-Tyr conjugate was dissolved in PBS, and mixed with trastuzumab, HRP and H2O2 solutions to prepare the HA-Tyr hydrogels. The final concentrations of HA-Tyr, trastuzumab, HRP and H2O2 were 1.75 wt.%, 0.3 mg/ml, 0.124 unit/ml and 437 respectively. For hydrogels incorporating hyaluronidase, 2.5 or 5 µl of 1000 unit/ml hyaluronidase was added into 1 ml of mixture solutions to achieve a final concentration of 2.5 or 5 unit/ml respectively. Rheological measurements of the hydrogels were performed with a HAAKE Rheoscope 1 rheometer (Karlsruhe, Germany) as described previously [25]. 2.3. Incorporation and release of trastuzumab from HA-Tyr hydrogels The mixture solution containing 1.75 wt.% HA-Tyr, 0.3 mg/ml trastuzumab, hyaluronidase of varying concentrations (0, 2.5 and 5 unit/ml), 0.124 unit/ml HRP abd 437 µM H2O2 were gently mixed by pipetting and then injected between two parallel glass plates clamped 1.5 mm apart. Gelation was allowed to proceed at 37oC for 2 h. Hydrogel disks with a diameter of 1.6 cm, were then cut from the hydrogel slab using a circular mold. To measure the percentage of 3
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trastuzumab that can be recovered from hydrogels, the disks were immersed in 3 ml PBS buffer containing 200 unit/ml hyaluronidase. Hydrogels were degraded overnight at 37oC, and trastuzumab concentration was measured by human IgG enzyme-linked immunosorbent assay (ELISA). The percentage of trastuzumab retrieved from hydrogels was then calculated. For cumulative release studies, hydrogel disks (1.6 cm in diameter, 1.5 mm in thickness) were placed in a plastic net and immersed in 20 ml of PBS buffer containing 0.05% sodium azide and 0.5% bovine serum albumin (BSA). At selected time points, 200 l of the solution was withdrawn and replaced with an equal volume of fresh buffer solution to maintain a constant total volume. The collected samples were stored in LoBind tubes (Eppendorf, Germany) at 4oC until measurement with a Human IgG ELISA kit.
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2.4. Degradation of hydrogel in vitro and in vivo For the degradation of hydrogels in vitro, hydrogel disks were prepared according to the protocols described in section 2.3. Each disk was placed in a plastic net and immersed in 20 ml PBS buffer containing 0.05% sodium azide and 0.5% BSA. At selected time points, the hydrogel disks were taken out of solution and excess water was soaked away with tissue paper. Then gel disks were weighed and returned to the buffer solution. For the degradation of hydrogels in vivo, 200 µl of mixture solution containing 1.75% HATyr, 0.63 mg/ml trastuzumab, hyaluronidase of varying concentrations (0, 2.5 and 5 unit/ml), 0.124 unit/ml HRP and 437 µM H2O2 were injected into NCr-Fixb1nu (NCr) mice subcutaneously. After 1, 3, 6, 14, 28 days, the remaining hydrogels were taken out and weighed.
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2.5. Inhibition of cell proliferation with HA-Tyr hydrogels that incorporated trastuzumab and hyaluronidase Five hundred microliters of BT-474 cell suspension containing 30,000 cells were seeded in a 24-well plate and incubated overnight. Then the hydrogels were prepared in 24-well cell culture inserts, in which 50 l of mixture solution containing 1.75 wt.% HA-Tyr conjugate, 0.05 mg/ml trastuzumab, hyaluronidase of different concentrations (0, 2.5 and 5 unit/ml), 124 unit/ml HRP and 437 µM H2O2 were added in each insert. After 2 h, the hydrogel-loaded inserts were placed above the cell-seeded 24-well plates, and an additional 500 l of culture media was added into the insert. After 1, 2, 3 and 4 days of incubation, cell viability was measured according to the protocols of alamarBlue assay. Fluorescence measurement was performed with the Infinite M200 (Tecan, Switzerland). Excitation and emission wavelengths were set at 545 and 590 nm respectively. The results were expressed as a percentage of viability compared with untreated cells. 2.6. Inhibition of tumor growth with HA-Tyr hydrogels that incorporated trastuzumab and hyaluronidase Two hundred microliters of BT-474 cells (5 107 cells/ml) suspended in Matrigel (BD, USA) were subcutaneously injected to the backs of 6-week-old female NCr-Fixb1nu (NCr) mice that were implanted with 17β-estradiol (0.72 mg/pellet, Innovative Research of America, USA). Fourteen days later when diameter of the tumor reached 5-10 mm, the mice were divided into 5 groups (n = 7) so that the average tumor size in each group was similar. Each mouse (~25 g) received a single-dose subcutaneous injection of 200 µl mixture solutions containing 1.75 wt.% HA-Tyr conjugate, 0.63 mg/ml trastuzumab, 0.124 unit/ml HRP, hyaluronidase of varying concentration (0, 2.5 and 5 unit/ml), 0.124 unit/ml HRP and 473 µM H 2O2. Also, single-dose 4
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subcutaneous injections of 200 l of PBS or 0.63 mg/ml trastuzumab solution were performed as a comparison. The dose of trastuzumab was maintained at 5 mg/kg for all the test groups. The site of injection was at least 2 cm away from the tumor. Tumors were measured with a digital caliper, and the tumor volumes (mm3) were calculated from the formula: volume = (length × width2)/2 [26]. On day 22, mice were euthanized, and then tumors were resected and fixed in 4 % formalin solution. In another experiment, we assessed the effect of the hydrogel without drugs on tumor growth by subcutaneous injection of mixture solutions containing all the components but no trastuzumab. A subcutaneous injection of mixture solution containing 0.63 mg/ml trastuzumab and 5 unit/ml hyaluronidase was also performed to assess the combination effect of trastuzumab and hyaluronidase on tumor growth. The care and use of laboratory animals were performed according to the approved protocols of the Institutional Animal Care and Use Committee (IACUC) at the Biological Resource Center (BRC) in Biopolis, Singapore.
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2.7. Histology and immunohistochemistry Tumor tissues were collected, fixed and stained by hematoxylin and eosin (H&E), Ki67 antibodies, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) as described previously [25]. The tissue slides were then examined with an Olympus microscope IX71. For Ki67 staining and TUNEL assay, three representative images were acquired from each group and the percentage of positive cells were quantified.
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2.8. Pharmacokinetics of trastuzumab NCr mice of ~25 g weight were divided into two groups (n = 3). The hydrogel group received the subcutaneous injection of 200 µl mixture solution described in section 2.6. The trastuzumab solution group received a subcutaneous injection of 0.63 mg/ml trastuzumab solution in PBS (200 µl). At selected time points, 20 µl of blood was taken from the tail vein of the mice, mixed with 3 µl of 37 mg/ml sodium citrate, and then centrifuged at 4oC, 3000 g for 5 min to collect the plasma. The plasma concentration of trastuzumab was measured by a human IgG ELISA kit. A PKSolver was used to calculate the pharmacokinetic parameters [27]. 2.9. Tissue reaction Balb/c mice aged between 5-6 weeks old were divided into two groups for the injection of hyaluronidase-incorporated hydrogels (0 and 5 unit/ml) respectively (n = 3). They received the subcutaneous injection of mixture solution described in section 2.6. At selected time points, mice were euthanized and the skin tissues, together with any remaining hydrogels, were collected. The tissues were then fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned for H&E staining. The tissue slides were then examined with an Olympus microscope IX71. 2.10. Statistical analyses Tumor size data in animal experiments are expressed as mean ± standard error of the mean. All other data are expressed as mean ± standard deviation. Statistical significance between two groups was determined by one-way ANOVA and student’s t-test. P 0.05), suggesting that the incorporation of hyaluronidase did not alter the mechanical properties of hydrogels. The time when the crossover of G’ and loss moduli (G’’) occurred (tgel point), and the time when G’ reached plateau (tplateau), were around 1 minute and 9 minutes for all three hydrogels respectively. The rapid gelation of these hydrogels would prevent an uncontrolled diffusion of the mixture solution during the gelation. Table 1 Characterization of trastuzumab-incorporated HA-Tyr hydrogelsa Sample HA-Tyr Trastuzumab Hyaluronidase tgel point tplateau G’ (wt.%l) (mg/ml) (U/ml) (min) (min) (Pa) HA-Tyr-TZB-0 1.75 0.3 0 0.93 ± 0.15 8.97 ± 0.32 500 ± 30 HA-Tyr-TZB-2.5 1.75 0.3 2.5 0.95 ± 0.21b 8.85 ± 0.49b 476 ± 43b HA-Tyr-TZB-5 1.75 0.3 5 1.07 ± 0.08c 8.80 ± 0.14c 452 ± 19c Note: Abbreviations; tgel point is determined as the time at which the crossover of storage modulus (G’) and loss modulus (G’’) occurred, tplateau refers to the time when G’ reaches plateau. a All hydrogels were formed with 437 µM of H2O2 and 0.124 units/ml of HRP. Results are shown as the mean values ± standard deviation (n = 3). b tgel point , tplateau and G’ of HA-Tyr-TZB-2.5 were not significantly different from those of HA-Tyr-TZB-0 (P>0.05). c tgel point , tplateau and G’ of HA-Tyr-TZB-5 were not significantly different from those of HA-Tyr-TZB-0 and HA-Tyr-TZB-2.5 (P>0.05).
3.2. Release of trastuzumab from HA-Tyr hydrogels that incorporated trastuzumab and hyaluronidase We utilized the human IgG enzyme-linked immunosorbent assay (ELISA) to measure the concentrations of trastuzumab. As ELISA detects protein through epitope recognition, we could detect and measure intact trastuzumab with this assay. To estimate the recovery of trastuzumab
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incorporated in HA-Tyr hydrogels, we treated the freshly-prepared hydrogels with 200 unit/ml hyaluronidase followed by ELISA measurement. We found that more than 90% of trastuzumab was retrieved from all the hydrogels regardless of the concentration of hyaluronidase incorporated in hydrogels (Fig.2), suggesting that the intactness of trastuzumab was well maintained during the crosslinking reactions.
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Then, we proceeded to examine the release profiles of trastuzumab from these hydrogels. For HA-Tyr-TZB-0, an initial release of trastuzumab was observed in the first two days, and afterwards no more release occurred (Fig. 3a). The incomplete release of trastuzumab in HA-TyrTZB-0 was due to the electrostatic interactions between trastuzumab and carboxyl groups of HATyr. Trastuzumab is a positively-charged protein and has been reported to interact with anionic polymeric nanoparticles [28]. In fact, we observed that with the increasing ionic strength of release buffer, the release of trastuzumab significantly increased, suggesting the electrostatic interactions between trastuzumab and HA-Tyr chains in the hydrogels (Fig. S1). We observed a continuous release of trastuzumab from HA-Tyr-TZB-2.5 and HA-Tyr-TZB-5 over 4 weeks. The release of trastuzumab from HA-Tyr-TZB-5 was faster than that from HA-Tyr-TZB-2.5. As hyaluronidase was incorporated in HA-Tyr-TZB-2.5 and HA-Tyr-TZB-5, the continuous release of trastuzumab from these hydrogels was considered to be triggered by the auto-degradation of the hydrogels.
Fig. 2. Percentage of trastuzumab retrieved from freshly-prepared HA-Tyr hydrogels that incorporated both trastuzumab and hyaluronidase (0, 2.5 and 5 unit/ml). a)
b)
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Fig. 3. (a) Cumulative release of trastuzumab from HA-Tyr hydrogels that incorporated both trastuzumab and hyaluronidase; (b) Degradation of hyaluronidase-incorporated HA-Tyr hydrogels in vitro.
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3.3. Degradation profiles of HA-Tyr hydrogels that incorporated trastuzumab and hyaluronidase We investigated the degradation profiles of HA-Tyr hydrogels that incorporated trastuzumab and hyaluronidase (0, 2.5 and 5 unit/ml). For HA-Tyr-TZB-0, we observed an initial increase of hydrogel weight during the first two days (Fig. 3b). Then, the weight of hydrogel reached a plateau, suggesting that the hydrogel was stable in phosphate buffer. In comparison, the weight of HA-Tyr-TZB-2.5 and HA-Tyr-TZB-5, after an initial increase, decreased continuously, suggesting that the hydrogels gradually degraded (Fig. 3b). Importantly, the rate of weight decrease of HA-Tyr-TZB-5 was faster than that of HA-Tyr-TZB-2.5, indicating that the rate of degradation of hydrogels was controlled by the concentrations of hyaluronidase incorporated in the hydrogels.
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We also analyzed the degradation and protein release when the same amount of hyaluronidase (2.5 units or 5 units per 1 ml gel) was added to the outside of HA-Tyr-TBZ-0 (e.g. in the release buffer). The degradation rate of the hydrogels was faster compared to hydrogel counterparts when hyaluronidase was incorporated in the hydrogel. Also, no further release of trastuzumab was observed after 2 weeks (Fig. S2). These results suggested that the incorporation of hyaluronidase in the hydrogel would be essential for the sustained release of trastuzumab.
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3.4. Inhibition of BT-474 cell proliferation with HA-Tyr hydrogels that incorporated trastuzumab and hyaluronidase in vitro
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We examined in vitro the anti-cancer efficacy of trastuzumab-incorporated HA-Tyr hydrogels with a BT-474 breast cancer cell line, which overexpresses human epidermal growth factor-2 (HER-2) receptor [29]. First, we assessed the effects of HA-Tyr hydrogels that incorporated hyaluronidase but no trastuzumab. The hydrogels with hyaluronidase (0, 2.5 and 5 unit/ml) did not show any significant difference in affecting the cell proliferation from day 1 to day 4 (Fig. 4 and Fig. S3), suggesting that the HA-Tyr hydrogel, hyaluronidase and the degradation compounds of the hydrogel did not affect the cell growth. Then, we examined the anti-proliferation effects of HA-Tyr hydrogels that incorporated both trastuzumab and hyaluronidase. The anti-proliferation effect increased with the increasing incubation time from day 1 to day 4. The difference in cell proliferation that was induced by hydrogels incorporating hyaluronidase (0, 2.5 and 5 unit/ml) was significant on day 4 (Fig. S3). The HA-Tyr hydrogels that incorporated 5 unit/ml hyaluronidase inhibited the proliferation of BT-474 cells more significantly than the hydrogels incorporating 0 or 2.5 unit/ml hyaluronidase (P
