Effect of freeze-drying on stability, thermo-responsive characteristics, and in vivo wound healing of erythropoietin-loaded trimethyl chitosan/glycerophosphate hydrogel
Erythropoietin (EPO) was successfully incorporated into a bioadhesive thermosensitive hydrogel based on trimethyl chitosan (TMC)/β-glycerophosphate (GP) for prevention and treatment of oral mucositis in cancerous patients. The aim of the present study was to evaluate the effect of freeze drying on thermo-responsive property of the hydrogel and structural stability of the loaded protein. The characteristics of the freeze-dried EPO-loaded hydrogel were determined by various methods: The gelation property by rheological analysis, aggregation of EPO in the formulation by SDS-PAGE, the protein secondary structure by far ultraviolet-circular dichroism (CD), and the antigenicity activity of EPO with ELISA techniques. The healing effect of the freeze-dried formulation was also investigated in vivo in Sprague-Dawley rats with chemotherapy-induced mucositis and compared with freshly prepared mixture. Finally, the retention time of the gel in the oral cavity was investigated in healthy volunteers. SDS-PAGE, CD, and ELISA confirmed the stability of conformational structure of loaded and released EPO. Severity of mucositis was markedly reduced in animals treated with freeze-dried EPO hydrogel, whereas the group received normal saline did not showed any significant healing effect. EPO salvia level was decreased rapidly using EPO solution compared to the gel. Approximately 40% of EPO was maintained on the buccal area in patients received the hydrogel system after 30 min. Therefore, the TMC/GP could preserve EPO stability after freeze drying and has the potential to be used in the treatment of OM and other oral or subcutaneous wounds.
Recombinant human erythropoietin (EPO) is approved by the US Food and Drug Administration (FDA) for treatment of anemia in patients with chronic kidney diseases and chemotherapy-associated anemia (1). A growing number of works indicates that EPO also provides therapeutic benefits other than its role in correcting anemia. It effectively inhibits the production of pro-inflammatory cytokines and also reduces the production of regenerative oxidative species (ROS) and membrane lipid peroxidase (2, 3). Some studies have demonstrated the positive effects of EPO in treatment of acute and chronic skin wounds and burns in animals and human (3-5). For instances, Galeano et al reported that subcutaneous administration of EPO in mice with deep-dermal second degree burn increased burn wound re-epithelialization and reduced the time to final wound closure (4). In another animal wound-healing model, topical treatment of the wounds of diabetic rats with EPO-containing cream decreased the extent of apoptosis and the areas of the open wound (6). Ferri et al reported that systemic EPO treatment is beneficial in cutaneous ulcers in patients with systemic ulceration (5). Recently we developed a novel thermosensitive hydrogel containing EPO for prevention and treatment of oral mocositis (OM) (7). OM is inflammation of oral mucosa of cancer patients as a result of radiotherapy and/or chemotherapy (8). The prevalence of OM is estimated 40% in patients receiving chemotherapy, 75% in those exposed to high dose chemotherapy and more than 90% in patients radiated for head and neck cancer (9). Our developed formulation successfully decreased the severity of OM in animal chemotherapy-induced OM model (7). Thermosensitive hydrogels exhibit solution to gel transition when the temperature changes from ambient to physiological. These hydrogels can therefore be conveniently administered as solution which swell and turn to gel relatively shortly after their application. The so formed gel will have the advantage of prolonged drug release in the surrounding medium; this improves the patient compliance owing to the lesser frequency of medication administration. However, hydrogels do not have enough stability in liquid form and they gradually convert to semisolid in room temperature and even in refrigerator. In the current study we aimed to evaluate the effect of freeze drying on thermo-responsive property of the hydrogel and conformational structural stability of the loaded EPO. The characteristics of the freeze-dried EPO-loaded hydrogel were determined by various methods: the gelation property by rheological analysis, aggregation of EPO in the formulation by SDS-PAGE, the protein secondary structure by far ultraviolet-circular dichroism (CD), and the antigenicity activity of EPO with ELISA techniques. The healing effect of the freeze-dried formulation was also investigated in vivo in Sprague-Dawley rats with chemotherapy-induced mucositis and compared with freshly prepared mixture. Finally, the retention time of the gel in the oral cavity was investigated in healthy volunteers.
Materials and methods
Trimethy chitosan (TMC) with degree of substitution 5% and molecular weight 150-300 kDa was synthesized in our laboratory. β-glycerophosphate disodium salt (GP) were purchased from Sigma Chemical Co. (St. Louis, MO).
Recombinant human erythropoietin (EPO) was obtained from Pasteur Institute (Tehran, Iran). Potassium dihydrogen phosphate, sodium hydroxide were supplied by Merck (Darmstads, Germany). Human EPO Platinum ELISA kit was supplied by eBioscience (Vienna, Austria).
Sprague-Dawley rats (5–6 weeks old, 200–250 g body weight) were obtained from the laboratory animal center of the Faculty of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Science, Isfahan, Iran. All animal experiments were carried out in accordance with Guide for the Care and Use of Laboratory Animals provided by the National Institute of Health and obtained the approval of The Research Ethics Committee of Isfahan University of Medical Science (approval code 294189).
Preparation of TMC/GP solution loaded with EPO
EPO solutions of 300 IU/mL was mixed with TMC solution. GP solution was then added to the mixture to obtain EPO-loaded hydrogel comprising final concentrations of TMC (5%) and GP (20%) (7).
Rheological behavior studies
The rheological behavior of EPO-loaded TMC/GP system was investigated by studying the viscosity as functions of temperature and time in a digital rotary viscometer (RVDV-III U, Daiki Sciences Co. USA) as described previously (7).
The mucoadhesion measurement was performed at 37 °C by means of a tensile stress tester (TA. XT plus Texture Analyzer, Stable Micro system, Godalming, UK). One hundred milligrams of EPO-loaded TMC/GP mixture was applied on a filter paper disc (area = 2 cm2) and fixed on the movable carriage of the apparatus. Cow buccal mucosa as biological substrate was hydrated with 100 μL of phosphate buffer at pH 6.8 and fixed on the sample holder. The formulation and the biological substrate were put in contact at a preload of 2500 mN for 5 min to allow the formation of the mucoadhesive joints. The preload was then removed and the movable carriage was moved upward at a prefixed speed of 2 mm/min up to the complete separation of the two surfaces. The force of detachment as a function of the displacement was recorded and the parameter work of adhesion (mN mm) (AUC) was calculated as the area under the force versus displacement curve (7).
In vitro release studies of EPO from the hydrogel
1 mL of the hydrogel solution containing EPO was placed in 25- mL flat-bottomed glass beakers and allowed to gel in an incubator at 37 °C for 5 min. Then, 30 mL phosphate buffer solution (PBS 0.1M, pH 6.8) was poured on the surface of gels and the vessels were shaken in a water bath shaker at 40 rpm and 37 °C. Aliquots of 1-mL samples from the supernatant solution were withdrawn at pre-determined time points. The same volume of fresh buffer was used to replace each sample immediately (7). EPO in the supernatant solution was assayed using human EPO Platinum ELISA kit, as directed by the manufacturer.
Effect of Freeze-drying on thermo-responsive, mucoadhesive, and EPO release properties of EPO-loaded TMC/GPsystem
The freeze-fried samples were prepared by immerging a polytetrafluoroethylene tube containing EPO-loaded TMC/GP solution into liquid nitrogen for several minutes to let them freeze, and then the frozen sample was lyophilized by a freeze drier (Christ Alpha 4.2 LD over, Germany) at −40 °C and a pressure of 0.4 bar for about 48 h. To study the thermo-responsive behavior, the powder of the dried polymer was first equilibrated in deionized water at 4 ºC, and then temperature was increased to 50 °C. The bioadhesive property and the release rate of EPO was also examined as described earlier.
Conformation investigation of EPO in the formulation using Far-UV CD Spectroscopy
The secondary structure of EPO released from the hydrogel was investigated by CD spectroscopy. The CD spectra were measured at room temperature using a J-810 spectropolarimeter from Jasco, Inc. (Easton, MD). The conditions of analysis were as follows: cell length, 1 cm; scanning speed, 100 nm/min; data pitch, 0.5 nm; sensitivity, standard; band width, 1 nm; wavelength range, 190–300 nm (far UV). CD pattern was compared with CD pattern of standard EPO set in default in equipment software database.
EPO antigenicity measurements
ELISA assay was used for estimating EPO antigenicity after 30 min exposure of EPO-loaded hydrogel in release medium. The incubation temperature and release medium were the same as those used in the experiment for EPO in vitro release study. Thirty min after incubation, the incubation medium was withdrawn and analyzed for antigenicity activity of the protein using EPO ELISA kit.
EPO integrity assessment using SDS–PAGE
The structural integrity andpossible aggregation of EPO following loadingand freeze drying processes were investigated by SDS–PAGE. An aliquot of the samples (EPO released from freeze-dried and freshly prepared samples during the first hour of in vitro studies) and several molecular mass reference standards
(14400–116000 Da, Thermo Scientific protein marker 26611) including lysozyme (14400), β-lactoglobulin (18400), restriction endonuclease (25000), lactate dehydrogenase (35000), ovoalbumin (45000), bovine serum albumin (66000) and β-galactosidase (116000) were electrophoresed on a 12% SDS polyacrylamide gel for 3 h at 100 V under nonreducing condition (without using any denaturants in the sample buffer). Proteins were visualized using Comassic blue staining. Standard EPO was used as control.
Healing effect of freeze-dried EPO-loaded hydrogel on Chemotherapy-induced OM in rats
OM was induced in Sprague-Dawley rats following three intraperitoneal injections of 5-flurouracil (5-FU) based on the protocol described in our previous study (7). The maximum severity of OM developed in all animals on days 7 to 9 after the initial injection. The OM-induced rats were randomly divided into three groups (5 subjects in each group). Group 1 served as negative control and received normal saline. In the beginning on day 6 after 5-FU injection, 1 mL of freshly prepared EPO-loaded hydrogel or freeze-dried sample was applied two times per day to cheek pouch of the rats in the groups 2 and 3, respectively. Four rats received no 5-Fu and served as normal control group. The treatment protocol was continued up to day 16. Three rats from each group were randomly selected and killed on days 12 for histopathological studies.
In vivo studies on healthy volunteers
Approval for in vivo studies on healthy volunteers was obtained from the Ethics Committee of Isfahan University of Medical Science (Project number: 194231). Six healthy volunteers (20-23 years old), after the explanation of the experimental protocol, agreed to participate in the study and a written informed consent was obtained from each subject. All subjects were healthy on the basis of the story, physical examination, serum chemistry profile, complete blood count, and urine analysis. All subjects were abstained from smoking, eating, or drinking for 90 min after the application of formulations. The volunteers were divided into two groups. Group 1 received EPO-laded TMC/GP hydrogel and group 2 received EPO solution. Salivary samples were collected by adsorbing the saliva with cotton swaps on the buccal area at 5, 15, 30, and 45 min after application of the formulations, then the cotton swabs were placed in test tubes containing water. EPO concentrations in the samples were determined using EPO ELISA kit.
Effect of freeze drying on characteristics of EPO-loaded TMC/GP hydrogel
Fig 1a and b showed the viscosity versus temperature and time for the freshly prepared of EPO-loaded TMC/GP and freeze-dried samples, respectively. As shown in Fig 1a, TMC/GP solution began to transfer to gel state at 33 °C and the viscosity increased to 14000 cP at 37 °C during 3 min. Freeze drying procedure did not significantly change the gelation time and temperature (Fig 1b). The parameter work of adhesion of freeze-dried EPO-loaded TMC/GP system was calculated 11020 ± 278 mN.mm which indicated the bioadhesive property of TMC/GP mixture. In vitro release of EPO from TMC/GP system before and after freeze drying has been shown in Fig 2. In both of the formulations the drug content was completely released during 8 h and there was no significant change in the burst effect and the drug release content from hydrogel before and after lyophilization.
Far- UV CD spectroscopy
The conformational stability of in vitro released EPO from the freeze-dried hydrogel was assessed by CD. After 1 h of release, the medium was withdrawn, and analyzed by CD. The CD profile of EPO in the far UV range is shown in Fig 3. As it is evident from this figure, two minima around 207and 221 nm, whichis typical of predominant α-helical structure of EPO was unaffected by the formulation and freeze drying process and superimposed almost entirely to that of standard EPO profile. This suggested that the protein maintained its α-helical structure and hence it’s secondary structure and physical stability.
Fig 4 shows SDS–PAGE of standard EPO, EPO released from samples after freeze drying. No aggregation or degradation band or any extra band indicating difference in molecular weight was observed between the released, and standard EPO. Therefore, we concluded that freeze drying and powder storage did not affect the structural integrity of EPO.
EPO antigenicity measurements
The ability of EPO to bind to antibodies that recognize specific epitopes on the EPO was used to confirm the maintenance of its tertiary structure after freeze drying. ELISA results indicated that the antibody binding was unchanged after freeze drying (91% as relative to EPO content of freshly prepared hydrogel). However, during storage for six mouths the activity was decreased to 79% compared to freshly prepared hydrogel.
Comparison the therapeutic effect of hydrogel before and after freeze drying
Severe mucositis was developed 7 days after the initial injection of 5-FU. Severity of mucositis were markedly reduced in animals treated with EPO hydrogel (freshly prepared and freeze-dried samples), whereas the group received normal saline did not showed any significant healing effect on day 16. In histological sections (Fig. 5a–d), various dilated and engorged capillaries, extensive infiltration of inflammatory cells, and hemorrhage were seen after 12 day in untreated (fig 5a) in comparison with the intact healthy mucosa (fig 5d). However, significant re-epithelialization and wound repair was observed in both group treated by EPO hydrogel (fig 5 b and c). There was no significant difference between the groups received freshly prepared hydrogel and freeze-dried gel which indicated the stability of EPO during freeze drying procedure and storage for 6 months.
In vivo studies on healthy volunteers
The saliva levels of EPO at different times after application of EPO-loaded hydrogel and EPO solution are shown in Fig. 6. EPO released from both the gel and formulations reached the highest levels 5 min after application. EPO salvia level was decreased rapidly using EPO solution compared to the gel. Approximately, 40% of EPO were maintained on the buccal area of volunteers after 30 min in the group received the gel.
OM is inflammation condition of oral mucosa as result of chemotherapy and/or radiotherapy. About the etiology of OM, it is generally accepted that some cytotoxic drugs such as 5-FU and methotrexate have direct inhibitory effects on DNA replication and mucosal cell proliferation which result in mucosal atrophy, collagen breakdown and ulceration (9,10). It is supposed that the main initiating factor of mucositis is the generation of reactive oxygen species (ROS) during chemotherapy and radiation (9). Moreover, the activation and increasing levels of pro-inflammatory cytokines including interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF)-α seem to have a role in development of OM (11). EPO effectively inhibits the production of ROS and lipid peroxidase (3-5). We recently developed a novel thermosensitive mucoadhesive gel containing EPO for prevention and treatment of OM (7). This formulation is liquid at room temperature and easily applied in buccal area, while upon exposure to the body temperature it would be solidified into the high viscous mucoadhesive hydrogel to withstand the removal action of salvia. The hydrogel also reduced the release rate of EPO to avoid repeated administration and improve patient compliance. The freeze drying procedure did not hamper the thermo-responsive and mucoadhesive properties of the optimized formulation. Moreover, stability of EPO in the freeze-dried powder formulation was evaluated using CD, SDA-PAGE, and ELISA techniques. Changes in the secondary structure of a proteins are best monitored by CD spectroscopy. EPO is a glycoprotein composed of only α-helices (12). The CD spectroscopy revealed that the protein maintained its α-helical structure and hence it’s secondary structure and physical stability. ELISA assay depends on the reaction of a predominant protein with specific antibody to form a complex. Because the antigen–antibody reaction is specific, antibodies are an important reagent for immunological research and clinical diagnostics. The ELISA method has very high sensitivity and specificity (13). ELISA antigenicity was considered to be an appropriate means for detection of potential changes of its antigenicity and tertiary structure. Therefore, we assumed that formulation and freeze drying technique did not affect the structural integrity and conformational stability of EPO. The aim of local drug delivery into the oral cavity is to provide therapeutic concentration of the drug at the required site. Here, the volunteers easily applied the formulation and more than 40% of EPO was maintained in oral cavity after 30 min. In general our developed formulation also offer advantages over the solid systems which are attached to the mucosa providing an application all around the mouth and achieve uniform distribution of the drug. TMC also offers an advantage over solution because of its inherent antimicrobial property.
Results of in vitro and in vivo studies indicated that EPO=loaded in as excellent candidate for prevention and treatment of OM. SDS-page, ELISA, and confirmed the stability of conformational structure of loaded and released EPO. When compared to the solution formulation, in healthy volunteers, TMC gel higher drug concentrations for longer time in the oral cavity due to its bioadhesive property. TMC also offers an advantage over solution because of its inherent antimicrobial property.
Fig 1. Effect of temperature on the viscosities of (a) EPO-loaded TMC/GP and (b) freeze-dried EPO-loaded TMC/GP after 6 months storage
Fig 2. The in vitro release profile of EPO from TMC/GP hydrogel before and after freeze
Drying (mean values ± SD, n = 3).
Fig 3. Far-UV CD spectra of standard and in vitro released EPO from the hydrogel
Fig 4. SDS–PAGE analysis of EPO: M; protein marker, , line 1; freeze-dried EPO-loaded TMC/GP after 6 months storage, line 2; freeze-dried EPO-loaded TMC/GP after 3 months storage, line 3; freeze-dried EPO-loaded TMC/GP after 1 months storage, line 4; freeze-dried EPO-loaded TMC/GP after 1 week and line 5; standard EPO. The arrow indicates EPO with approximately molecular weight of 30000 Da.
Fig 5. Histological appearance of chemotherapy-induced oral mucositis in rats on day 12 (a) negative control group received normal saline (b) group 2 received freshly prepared EPO-loaded hydrogel (c) group 3 received freeze-dried EPO-containing formulation (d) normal control group received no 5-Fu. All evaluations were performed on hematoxylin and eosin routine staining (× 400)
Fig 6. Relative remaining of EPO in buccal area of healthy volunteers at different times (mean values ± SD, n = 3)
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