materials Article
Floating Hydrogel with Self-Generating Micro-Bubbles for Intravesical Instillation Tingsheng Lin 1,† , Xiaozhi Zhao 1,† , Yifan Zhang 2 , Huibo Lian 1 , Junlong Zhuang 1 , Qing Zhang 1 , Wei Chen 1 , Wei Wang 1 , Guangxiang Liu 1 , Suhan Guo 1 , Jinhui Wu 2, *, Yiqiao Hu 2, * and Hongqian Guo 1, * 1
2
*
†
Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, China;
[email protected] (T.L.);
[email protected] (X.Z.);
[email protected] (H.L.);
[email protected] (J.Z.);
[email protected] (Q.Z.);
[email protected] (W.C.);
[email protected] (W.W.);
[email protected] (G.L.);
[email protected] (S.G.) State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China;
[email protected] Correspondence:
[email protected] (J.W.);
[email protected] (Y.H.);
[email protected] (H.G.); Tel.: +86-139-1302-6062 (J.W.); +86-136-0140-2829 (Y.H.); +86-136-0517-1690 (H.G.); Fax: +86-25-83596143 (J.W. & Y.H.) These authors contributed equally to the work.
Academic Editor: Franz E. Weber Received: 7 November 2016; Accepted: 5 December 2016; Published: 12 December 2016
Abstract: Intravesical instillation is the main therapy for bladder cancer and interstitial cystitis. However, most drug solutions are eliminated from bladder after the first voiding of urine. To solve this problem, we proposed a floating hydrogel with self-generating micro-bubbles as a new delivery system. It floated in urine, avoiding the urinary obstruction and bladder irritation that ordinary hydrogels caused. In this study, we abandoned traditional gas-producing method like chemical decomposition of NaHCO3 , and used the foamability of Poloxamer 407 (P407) instead. Through simple shaking (just like shaking SonoVue for contrast-enhanced ultrasound in clinical), the P407 solution will “lock” many micro-bubbles and float in urine as quickly and steadily as other gas producing materials. In vivo release experiments showed that drug was released continually from hydrogel for 10 h during the erosion process. Thus, the residence time of drug in bladder was prolonged and drug efficacy was improved. In vivo efficacy study using rabbit acute bladder injury model showed that prolonged drug residence time in bladder increased the efficiency of heparin in the protection of bladder mucosal permeability. Therefore, our floating hydrogel system with self-generating micro-bubbles was single-component, simply prepared and efficacy enhancing, successfully exempting users from worries on safety and clinical efficiency from bench to bedside. Keywords: floating hydrogel; shaking; thermo-sensitive hydrogel; intravesical instillation; acute bladder injury
1. Introduction Diseases of the urinary bladder account for a large number of severe medical conditions affecting patients of all ages. The major diseases affecting the urinary bladder are bladder cancer and intractable interstitial cystitis. Intravesical instillation is the most common treatment for the bladder cancer and intractable interstitial cystitis [1]. It is performed by instilling chemical drugs to bladder using a urethral catheter. The efficacy of the intravesical treatment depends on the residence time of drug inside the bladder, which relates to the adhesive capability of the drug onto the urinary bladder wall [2]. However, most drug solutions were eliminated during the first voiding of urine after intravesical Materials 2016, 9, 1005; doi:10.3390/ma9121005
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instillation. The efficacy of intravesical instillation relies on the drug residence time in bladder, so it is imperative to develop a controlled drug delivery system. Recently, hydrogel had been used as drug reservoir to extend the drug residence time in bladder [3–5]. However, due to the high viscosity of hydrogel, one serious problem is urinary obstruction. Gels adhering to the bladder may block the urinary tract, such as internal urethral orifice and ureteric orifice with narrow opening. As hydrogels attach to the bladder wall, another problem is serious bladder irritation symptoms, such as blood clots in bladder [6]. The two floating hydrogel drug delivery systems we developed before were able to overcome these two shortcomings and prolonged the resident time of drug in the bladder [7,8]. The first floating hydrogel system consisted of thermo-sensitive polymer (Poloxamer 407) and NaHCO3 , which was liquid at low temperature while forming gel at body temperature (37 ◦ C). In the presence of H+ , NaHCO3 decomposed and produced CO2 , which attached to the surface of hydrogel and float the hydrogel in urine. Hence, the urinary tract will not be blocked, and the encapsulated drug released in a controlled manner. However, we need to acidify the urine to provide enough acid environment (pH < 5.6) for hydrogel floating in urine. To realize urine acidification, patients have to take oral drugs, like ammonium chloride and vitamin C. Extra drugs increase the risk of side effects including acid–base imbalance and electrolyte disturbances. Thus, the practical application of this floating hydrogel system was limited. In the second floating hydrogel system, NaHCO3 was substituted by Ammonium bicarbonate (NH4 HCO3 ) as micro-bubbles producers. NH4 HCO3 decomposed at body temperature without acidifying the urine and generated micro-bubbles inside hydrogel, which also float the hydrogel in urine. However, NH4 HCO3 is unstable and easily decomposed, especially in the liquid solution, so this floating hydrogel system could not be stored for a long time. In addition, both NaHCO3 and NH4 HCO3 are inorganic compounds, which might react with drugs in floating hydrogel and lead to chemical change of drugs. Therefore, more ideal floating scheme is required for the floating hydrogel system in practical clinical application. In this study, we developed a floating hydrogel drug delivery system merely with Poloxamer 407, with no additive for producing micro-bubbles. In fact, P407 solution could produce many micro-bubbles through shaking, stirring, or homogenizing, as P407 is a non-ionic surfactant [9]. In this system, no extra conditions, such as specific temperature and pH, were required; hence, safety and efficiency problems from bench to bedside were solved radically. By just shaking the hydrogel solution (like shaking SonoVue for contrast-enhanced ultrasound in clinical), micro-bubbles can be viewed as gas-filled micelles formed by surfactant molecules, whose hydrophobic tail groups face the hydrophobic gas and hydrophilic head groups face the aqueous phase [10,11]. Due to the viscosity of P407 solution, micro-bubbles were suspended for a certain amount of time and did not escape easily. Moreover, the micro-bubbles could be “locked” in hydrogel when the P407 solution converted to gel state at higher temperature due to its thermo-sensitive property (Figure 1). The P407 solution after shaking converted to gel and float in urine immediately after injected into bladder, and the encapsulated drug was released in a controlled manner. Our study showed that this floating hydrogel drug delivery system was easily prepared and safe. Because the production of micro-bubbles was milder and slower, the residence time of drug was extended and efficacy of intravesical drug delivery was improved.
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Figure1.1. The The schematic schematicdiagram diagramof offloating floatinghydrogel hydrogelpreparation preparationby byshaking. shaking. Figure
2. Materials and Methods 2. Materials and Methods 2.1. Materials Materials 2.1. Poloxamer407 407(P407) (P407) was was purchased purchased from from Meilun Meilun Biology Biology Technology Technology Company, Company,Ltd. Ltd. (Dalian, (Dalian, Poloxamer China).Rhodamine Rhodamine B (RhB), Protamine sulfate and heparin were purchased from China). B (RhB), Protamine sulfate (PS) and (PS) heparin were purchased from Sigma-Aldrich Sigma-Aldrich (St. Louis, MO, USA). Thirty New Zealand white male rabbits, weighting on average (St. Louis, MO, USA). Thirty New Zealand white male rabbits, weighting on average 2000 g (range, 2000 g (range, 1800–2500 g),from were from the Experimental Animal Center, University of 1800–2500 g), were obtained theobtained Experimental Animal Center, University of Yangzhou, China. Yangzhou, China. All animal protocols were approved by Institutional Animal Care and Use All animal protocols were approved by Institutional Animal Care and Use Committee (IACUC) of Committee (IACUC) of Nanjing University. Nanjing University. 2.2. Preparation Preparationand andOptimization OptimizationofofFloating FloatingHydrogel Hydrogel 2.2. Thenon-floating non-floating hydrogel is mixture the mixture andwater, distilled water, the P407 The hydrogel is the of P407of andP407 distilled and the P407and concentration concentration 45% (w/v). To prove thatcould micro-bubbles could be produced solution and was 45% (w/v).was To prove that micro-bubbles be produced by P407 solution by andP407 reserved in P407 reserved in P407 solution, the P407 solutions was shaken by hand up and down at a speed of ≥2 solution, the P407 solutions was shaken by hand up and down at a speed of ≥2 round/s (Figure 1), ◦ ◦ round/s (Figure and37stored at 0 °C and 37 °C, respectively. and stored at 0 C1),and C, respectively. Todetermine determine the the volume volume change of hydrogel 1010 mL of To hydrogel in inrespect respectto tothe thedensity densityofofmicro-bubble, micro-bubble, mL P407 solution was shaken by hand for 0, 10, 20, 30, 40 times respectively. The volume changes of of P407 solution was shaken by hand for 0, 10, 20, 30, 40 times respectively. The volume changes hydrogels after effect of of environmental environmental of hydrogels afterdifferent differentshaking shakingtimes timeswere wererecorded. recorded. To To determine determine the effect factors and andshaking shaking force forceon onmicro-bubble micro-bubble generation, generation, the the volume volume change change of of hydrogel hydrogel in inrespect respectto to factors thedensity densityof ofmicro-bubble micro-bubblewas wasmeasured. measured. Ten Tenmilliliters millilitersof ofP407 P407solution solutionwas wasshaken shaken by byhand handfor for the 20times timesat atdifferent differentenvironmental environmentalfactors factorsand andthe thevolume volumechanges changesof ofhydrogels hydrogelsafter aftershaking shakingwere were 20 recorded.ToTo observe environmental gel solutions were atshaking the local different recorded. observe environmental effect,effect, gel solutions were shaking the localatdifferent temperature ◦ ◦ ◦ ◦ temperature (3.525.3 °C, 11.5 °C,31.9 25.3 C), °C and °C), local different humidity 46.8%, 69.4%local and (3.5 C, 11.5 C, C and local31.9 different humidity (22.6%, 46.8%,(22.6%, 69.4% and 88.2%), 88.2%), local different atmosphere pressure (1005/100Pa, Pa,1013/100 1009/100 Pa Pa,and 1013/100 Pa and Pa). different atmosphere pressure (1005/100 Pa, 1009/100 1015/100 Pa).1015/100 To evaluate To evaluate shaking force on micro-bubble generation, shaking speed in respect to the shaking force shaking force on micro-bubble generation, shaking speed in respect to the shaking force was measured. was measured. Shaking defined asmove the time of hand and(hand downmove in one (hand Shaking speed defined as speed the time of hand up and downmove in oneup cycle upcycle and down move upcycle, and down was one theor distance of hand up or down was 50 cm). was one the distance of cycle, hand up down was 50 cm). Toevaluate evaluatethe theeffect effectof ofshaking shakingtime timeon onmicro-bubbles micro-bubblesamount amountin inhydrogel, hydrogel,P407 P407solution solutionwas was To shakenby byhand handfor for0, 0,10, 10,20, 20,30, 30,40 40times timesrespectively. respectively.Micro-bubbles Micro-bubblesproduced producedin inhydrogel hydrogelsolution solution shaken after different shaking time were recorded with photos and detected by ultrasound (B/K, after different shaking time were recorded with photos and detected by ultrasound (B/K, Copenhagen, Copenhagen, To count number of micro-bubbles, hydrogel was slide put on a glass slide Denmark). To Denmark). count the number of the micro-bubbles, hydrogel was put on a glass and observed anda observed via microscope a Zeiss M2Bio microscope (Zeiss,Germany). Oberkochen, The zoom was of microscope via Zeiss M2Bio (Zeiss, Oberkochen, TheGermany). zoom of microscope 40×, and was 40×, andcaptured. images were P407 shaking solutionwas afterput shaking put in °C water to observe its images were P407captured. solution after in 37 ◦was C water to37 observe its floating state. floating state.
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2.3. Characterizations of Floating Hydrogel To obtain information on the pore structure of hydrogels, the floating hydrogel shaken for 20 times was observed using Scanning electron microscopy. The samples were plunged in liquid nitrogen and freeze-dried to maintain the porous structure without any collapse. The samples were mounted on the base plate and coated with gold. The morphology was investigated using a Hitachi (Tokyo, Japan) S-570 Scanning Electron Microscope. The bubble size distribution of the floating hydrogel was investigated using electron microscope (Nikon, Tokyo, Japan). The floating hydrogel after 20-times shaking was put on a glass slide, the zoom of microscope was 40×. Three fields were randomly selected to calculate the bubble size distribution. The apparent viscosity was determined by a NDJ-1 viscometer (Shanghai Balance Instrument Factory, Shanghai, China). Twenty milliliters of hydrogel solution after 20-times shaking was put in a 25 mL beaker and placed in water bath. Then the solution was heated at a speed of 1 ◦ C/min and the viscosity was recorded. The erosion time was the time it takes for gel to dissolve completely in water. The volume of 5 mL, 10 mL, 15 mL, 20 mL floating hydrogels was injected into 37 ◦ C water, and the erosion time of floating hydrogels were recorded. The storage stability of the hydrogel of 45% P407 was also recorded. A digital timer (Thermo Fisher Scientific, Boston, MA, USA) was used for recording. 2.4. Incorporation of Drug in Floating Hydrogel To study the release of floating hydrogel in vitro and in vivo, Rhodamine B was incorporated into hydrogel. A calculated amount of P407 (45%) was successively dissolved in RhB solution at 4 ◦ C to form a RhB-loaded hydrogel solution. The concentration of RhB was 0.005% (w/v). To study the efficacy of floating hydrogel in acute bladder injury model, Heparin was incorporated into hydrogel. A calculated amount of P407 (45%) was successively dissolved in Heparin solution at 4 ◦ C to form a Heparin-loaded hydrogel solution. The concentration of Heparin was 1500 IU/mL. 2.5. Release Study In Vitro The release study was performed in the dissolution tester (ZRS-8G, Instruments of Tianjin University, Tianjin, China). The PBS (pH = 7, 200 mL) in the beaker of dissolution tester was heated to 37 ◦ C. RhB solution (50 ug/mL, 5 mL), RhB-loaded floating hydrogel (5 mL) and RhB-loaded non-floating hydrogel (5 mL) was injected into solution, respectively. At predetermined time points, 3 mL solution was collected and substituted with the same volume of fresh solution. The amount of RhB was determined by UV spectrophotometry (UV-2450, Shimadzu, Kyoto, Japan) at 555 nm. 2.6. Verification of Hydrogel Floating In Vivo Rabbit was anesthetized with pentobarbital (30 mg/kg, intravenous injection). Floating hydrogel solution was intravesically instilled into bladder using a catheter. The floating hydrogel in bladder was detected by B ultrasound. 2.7. Release Study In Vivo The rabbits were randomly chosen. They were maintained in a controlled atmosphere of 12 h dark/light cycle, 22 ± 2 ◦ C temperature and 50%–70% humidity, with free access to pellet feed and fresh tap water. The animals were supplied with dry food pellets commercially available. In fact, the gel solution was stored at 0 ◦ C condition before injected to the bladder and the viscosity of 45% P407 was about 200 mPa·s at this temperature. The viscosity was low. Therefore, it is easy to inject the gel solution from the catheter to bladder. Rabbits were intravesically instilled with 3 mL normal saline, free RhB solution, RhB-loaded floating hydrogel and RhB-loaded non-floating hydrogel solution respectively. The RhB -loaded non-floating hydrogel solution did not undergo shaking.
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All animals were fastened on a desk. Intravesical administration was accomplished using a catheter (9F) inserted into the bladder through the urethra. Urine samples were collected. The concentration of RhB was determined by a fluorescence microplate reader (Safire, TECAN or Molecular Devices M3, Männedorf, Switzerland). The frozen section was immediately prepared after isolating rabbit bladder tissues. The fluorescence in the bladder section was observed using a Zeiss M2Bio fluorescence microscope. The thickness of frozen biopsy was 10 µm, and the exposure time of fluorescence microscope was 1 s. 2.8. The Efficacy of Floating Hydrogel in Acute Bladder Injury Model Fifteen rabbits were randomly divided into five groups, with three rabbits for each group. All rabbits were fastened on the desk for intravesical instillation. For the control group, the bladders of rabbits were pretreated with 10 mL normal saline for 60 min, followed by 3 mL normal saline. For the PS + Saline group, the bladders of rabbits were pretreated with 10 mL Protamine sulfate (PS) (20 mg/mL) for 60 min, followed by 3 mL saline. For the PS + Gel group, the bladders of rabbits were pretreated with 10 mL PS (20 mg/mL) for 60 min, followed by 3 mL non-floating hydrogel (45% P407 hydrogel solution without shaking). For the PS + Heparin solution group, the bladders of rabbits were pretreated with 10 mL PS (20 mg/mL) for 60 min, followed by 3 mL heparin solution (1500 IU/mL). For the PS + Heparin loaded floating hydrogel group, the bladders of rabbits were pretreated with 10 mL PS (20 mg/mL) for 60 min, followed by 3 mL heparin loaded floating hydrogel (1500 IU/mL). After the treatments, all rabbits bladders were intravesically instilled 20 mL saline to trigger the first voiding of urine. After 10 h, all rabbits were intravesical instilled RhB solution (5 ug/mL) for 30 min. Then all rats were executed and bladder tissues were obtained. The frozen section was immediately prepared. The fluorescence in the bladder section was observed using a Zeiss M2Bio fluorescence microscope. The thickness of frozen biopsy was 5 µm, the exposure time of fluorescence microscope was 83 ms. The fluorescence depth of bladder tissues were calculated by the software Image-pro 6.0 (Media Cybernetics, Bethesda, MD, USA). 3. Results 3.1. Preparation and Optimization of Floating Hydrogel P407 was chosen as the matrix of floating hydrogel, and 45% (w/v) P407 was selected as optimal concentration according to our previous work [8]. As P407 was a kind of surfactant, the surfactant molecules could coat micro-bubbles by shaking, stirring, or homogenizing [9]. Enough micro-bubbles enlarge the volume of hydrogel and hence enhance the buoyancy, which eventually float the hydrogel. A large amount of micro-bubbles were produced in hydrogel solution after shaking for 40 times, while the hydrogel became opaque (Figure 2). To confirm that micro-bubbles were blocked in hydrogel at different temperature, hydrogel solutions after shaking were placed at 0 ◦ C and 37 ◦ C respectively. Results showed that the hydrogel could hold micro-bubbles in gel during the whole experiment at 37 ◦ C (Figure 2a), while micro-bubbles dissipated gradually at 0 ◦ C (Figure 2b). Because P407 is a thermo-sensitive material, it was gel state at 37 ◦ C, but liquid state at 0 ◦ C. Once the gel network formed, micro-bubbles was “locked” in hydrogel, and the hydrogel volume increased at the same time, so the buoyancy of hydrogel was increased and ensure the hydrogel stably float in liquid. We then investigate the effects of shaking time on generation of micro-bubbles. Hydrogel solution was shaken for 0, 10, 20, 30, 40 times respectively. Micro-bubbles produced in hydrogel solution were recorded with camera (Figure 3a). The density of micro-bubbles in gel increased with times of shaking. However, the hydrogel solution before shaking was transparent, with no micro-bubbles observed. To further observe the micro-bubbles produced in hydrogel solution, they were detected by ultrasound and images were captured (Figure 3b). As micro-bubbles in hydrogel was an ultrasound contrast agents, significant enhancement will be observed when a lot of micro-bubbles produced. The enhancement of hydrogel ultrasound images was also proportional to the shaking time. Similarly, the microscope images
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showed that the number of micro-bubbles in hydrogel increased with times of shaking (Figure 3c). According to these images, the mean number of micro-bubbles in hydrogel shaken for 10, 20, 30 and Materials 2016, 9, 1005 6 of 14 40 times were 49 ± 5,107 ± 15, 165 ± 8 and 250 ± 16, respectively (Figure S2). To determine the volume change of hydrogel in respect to the density of micro-bubble, 10 mL of P407 solution was of micro-bubbles in hydrogel shaken for 10, 20, 30 and 40 times were 49 ± 5,107 ± 15, 165 ± 8 and 250 ± shaken hand 0, 10, 20, 30, 40 times. the Thevolume volume changes of hydrogels after 16,by respectively (Figure S2).and To determine change of hydrogel in respect to different the densityshaking of timesmicro-bubble, were recorded. Theofmean change of hydrogel for and 10, 20, 30 and 40volume times were 10 mL P407 volume solution was shaken by hand 0,shaken 10, 20, 30, 40 times. The 0.47 ±changes 0.15, 1.23 0.25, 1.9 after ± 0.20, and 2.23 ± 0.25, respectively (FigureThe S6),mean hydrogel volume increased of ± hydrogels different shaking times were recorded. volume change of hydrogel shaken for 10, 20, 30 andhydrogel 40 times were 0.47 ± 0.15, 1.23 ± 0.25, 1.9To ± 0.20, and 2.23the ± 0.25, indicated air gas incorporated into and formed micro-bubbles. determine effect of respectivelyfactors (Figure S6),shaking hydrogel volume increased indicated air gasthe incorporated into hydrogel environmental and force on micro-bubble generation, volume change of hydrogel and formed To determine themeasured. effect of environmental factors and shaking force on in respect to the micro-bubbles. density of micro-bubble was Results showed that environmental factors micro-bubble generation, the volume change of hydrogel in respect to the density of micro-bubble (temperature, humidity, and atmosphere pressure) had no significant effect on micro-bubble generation was measured. Results showed that environmental factors (temperature, humidity, and atmosphere (Figure S7a–c). However, the hand shaking speed had great impact on micro-bubble generation of pressure) had no significant effect on micro-bubble generation (Figure S7a–c). However, the hand gel solution, shaking time greater than 1 s led to inadequate micro-bubbles generated for floating shaking speed had great impact on micro-bubble generation of gel solution, shaking time greater ◦ C water, these hydrogel solutions hydrogel To test micro-bubbles the floating state of hydrogel in 37hydrogel than (Figure 1 s led toS7d). inadequate generated for floating (Figure S7d). To test the ◦ were floating put intostate 37 of C hydrogel water. Results that hydrogel the groups of hydrogels 20 times floated in 37 °Cshowed water, these solutions were putshaken into 37over °C water. Results stablyshowed in water, groups hydrogel (not over shaken or shaken times) float (Figure thatother the groups of of hydrogels shaken 20 times floated10stably in failed water, to other groups of 3d). hydrogel (notand shaken or shaken 10 times)of failed to float were (Figure 3d). The erosion and The gelation The erosion time gelation temperature hydrogels also tested (Figuretime S1a,b). gelation ◦ C, 11.5 ◦ C, 11.7of ◦ C, temperature of hydrogels were 10, also20, tested (Figure S1a,b).was The11.5 gelation temperature hydrogels temperature of hydrogels shaken 30 and 40 times 11.9 ◦ C and 10, 20, 30 The and 40 times time was 11.5 °C, 11.5 °C, 11.7 °C, 11.9(not °C and 11.9 or °C,shaken respectively. The and 11.9 ◦shaken C, respectively. erosion of non-floating hydrogel shaken 10 times) erosion time of non-floating hydrogel (not shaken or shaken 10 times) and floating hydrogel (shaken floating hydrogel (shaken 20–40 times) were about 6.5 h and 6 h, respectively. The gelation temperature 20–40 times) were about 6.5 h and 6 h, respectively. The gelation temperature and erosion time of and erosion time of hydrogels showed no differences before and after shaking. These parameters hydrogels showed no differences before and after shaking. These parameters indicated that the indicated that the shaking did not affect the inherent property of hydrogel, but only floated it. shaking did not affect the inherent property of hydrogel, but only floated it.
Figure 2. Bubbles in hydrogel solutions after shaking at 0 °C and 37 °C. To confirm that
Figure 2. Bubbles in hydrogel solutions after shaking at 0 ◦ C and 37 ◦ C. To confirm that micro-bubbles micro-bubbles were blocked in hydrogel at different temperature, hydrogel solutions after shaking were blocked in hydrogel at37 different temperature, hydrogel solutions after shakinginwere placedthe at 0 ◦ C were placed at 0 °C and °C, respectively. The hydrogel could hold micro-bubbles gel during ◦ and 37 C, respectively. hydrogel could hold micro-bubbles in at gel0 during whole experiment atThe 37 °C (a). Micro-bubbles dissipated gradually °C (b). the whole experiment at 37 ◦ C (a). Micro-bubbles dissipated gradually at 0 ◦ C (b). 3.2. Characterization of Floating Hydrogel
3.2. Characterization Floatingmorphology Hydrogel was investigated by SEM (Figure 4a). The floating hydrogel The floatingofhydrogel was floating porous, with clear polymer strands. Micro-bubbles produced floating4a). hydrogel could be hydrogel seen The hydrogel morphology was investigated by SEMin(Figure The floating in gel networks. The size of micro-bubbles in hydrogel ranged from 20 μm to 400 μm, most were was porous, with clear polymer strands. Micro-bubbles produced in floating hydrogel could be seen 80~140 μm (Figure 4b). The viscosity of hydrogel increased drastically at 10.5 °C (its sol-gel in gel networks. The size of micro-bubbles in hydrogel ranged from 20 µm to 400 µm, most were transition temperature) and reached maximum at 12 °C (Figure 4c). To evaluate the effect of gel ◦ C (its sol-gel transition 80~140 µm (Figure 4b). The increased drastically 10.5 volume on hydrogels, weviscosity recorded of thehydrogel erosion time in aqueous solution.at The erosion time of floating ◦ C (Figure 4c). To evaluate the effect of gel volume on temperature) reached at 12 hydrogel and increased withmaximum the hydrogel volume, ranging from 6 to 24 h as the hydrogel volume hydrogels, wefrom recorded the20erosion time4d). in aqueous solution. erosion time floating increased 5 mL to mL (Figure The storage stability The of the hydrogel wasofalso tested.hydrogel The
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increased with the hydrogel volume, ranging from 6 to 24 h as the hydrogel volume increased from 2016, 9, 1005 4d). The storage stability of the hydrogel was also tested. The hydrogel 7 of 14 5 mLMaterials to 20 mL (Figure could be stored up to nine months without significant change of the gelation. The drug entrapment efficiency hydrogel could be stored up to nine months without significant change of the gelation. The drug Materials 2016, 1005gelation of hydrogel. 7 of 14 was 100% due to9,the entrapment efficiency was 100% due to the gelation of hydrogel. hydrogel could be stored up to nine months without significant change of the gelation. The drug entrapment efficiency was 100% due to the gelation of hydrogel.
Figure 3. Effects of shaking time on bubbles in hydrogel: (a) bubbles in hydrogel solution recorded
Figure 3. photos; Effects (b) of shaking on bubbles hydrogel: (a) bubbles hydrogel solutionunder recorded with bubbles intime hydrogel solutionin detected by ultrasound; (c)in bubbles in hydrogels with microscope; photos; (b) and bubbles in hydrogel solution detected by ultrasound; (c) bubbles in hydrogels under the floating of hydrogels. Figure 3. Effects(d) of shaking timestate on bubbles in hydrogel: (a) bubbles in hydrogel solution recorded microscope; and (d) the floating state of hydrogels. with photos; (b) bubbles in hydrogel solution detected by ultrasound; (c) bubbles in hydrogels under microscope; and (d) the floating state of hydrogels.
Figure 4. Characterizations of floating hydrogel: (a) the floating hydrogel morphology under SEM; (b) bubble size distribution of the floating hydrogel; (c) viscosity of the floating hydrogel; and (d) erosion time of the floating hydrogel with different(a) volume. Figure 4. Characterizations of floating hydrogel: the floating hydrogel morphology under SEM;
Figure 4. Characterizations of floating hydrogel: (a) the floating hydrogel morphology under SEM; (b) bubble size distribution of the floating hydrogel; (c) viscosity of the floating hydrogel; and (d) (b) size distribution 3.3.bubble Release Study In Vitro of the floating hydrogel; (c) viscosity of the floating hydrogel; and (d) erosion erosion time of the floating hydrogel with different volume. time of the floating hydrogel with different volume. The release profiles of free RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded 3.3. Release Study In floating hydrogel inVitro vitro were investigated (Figure 5). RhB served as fluorescence indicator. The
The release profiles of free RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded floating hydrogel in vitro were investigated (Figure 5). RhB served as fluorescence indicator. The
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3.3. Release Study In Vitro The release profiles of free RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded floating hydrogel in vitro were investigated (Figure 5). RhB served as fluorescence indicator. The curves Materials 2016, 9, 1005 8 of 14 depicted the cumulative release amount of RhB as a function of time. The control (free RhB) dispersed Materials 2016, 9, 1005 8 of 14 curves depicted thehomogenous cumulative release amount of RhBand as a the function of time. The control (free RhB) immediately to form solution in water, cumulative release reached 96.3% in curves depictedwhile, the to cumulative release amount of RhBin as a function time. The control (free RhB) dispersed immediately form homogenous solution water, andofthe cumulative release reached 10 min after injection, after injecting RhB-loaded non-floating hydrogel or RhB-loaded floating dispersed immediately to form homogenous solution in water, and the cumulative release hydrogel reached 96.3% in 10 min after injection, while, after injecting RhB-loaded non-floating or for hydrogel into water, floating hydrogel could float on the surface of water immediately. The time 96.3% in 10 minhydrogel after injection, while, floating after injecting RhB-loaded non-floating hydrogeloforwater RhB-loaded floating into water, hydrogel could float on the surface RhB to beRhB-loaded completely released from gelwater, in the non-floating hydrogel group and floating floating hydrogel into floating hydrogel could float on the surface of water hydrogel √ immediately. The time for RhB to be completely released from gel in the non-floating hydrogel group were 6.5 and 6 h, respectively. We also found that the Higuchi model (Qt = K t) could predict immediately. The time for RhB to be completely released from gel in the non-floatingHhydrogel group and floating hydrogel group were 6.5 and 6 h, respectively. We also found that the Higuchi group and floating hydrogel group were 6.5 and in, 6 h,which respectively. We also constant found that (K the Higuchi the drug release from floating hydrogel perfectly the release 39.9 and the H ) is modelmodel (Qt =(Qt KH=√t) predict the from floating floatinghydrogel hydrogel perfectly in, which KHcould predict thedrug drugrelease release from perfectly in, which the the √t) could correlation coefficient (R) is 0.99 [12], which indicated that drug release from floating hydrogel releaserelease constant (KH)(K isH39.9 and thethe correlation (R)isis0.99 0.99 [12], which indicated that drugwas a constant ) is 39.9 and correlationcoefficient coefficient (R) [12], which indicated that drug controlled-release process and square time dependent. releaserelease from from floating hydrogel was a root controlled-release processand and square root dependent. floating hydrogel was a controlled-release process square root timetime dependent.
Figure 5. Cumulative release of free-RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded
Figure 5. Cumulative release of free-RhB solution, RhB-loaded non-floating hydrogel and RhB-loaded floating hydrogel (Mean ± SD, n = 3). floating hydrogel (Mean release ± SD, of n =free-RhB 3). Figure 5. Cumulative solution, RhB-loaded non-floating hydrogel and RhB-loaded 3.4. Release Study (Mean In Vivo± SD, n = 3). floating hydrogel
3.4. Release Study In the Vivo Firstly, process of intravesical injection of floating hydrogel into rabbit bladder was detected 3.4. Release Study In Vivo by ultrasound (Figure 6). Catheter was inserted into rabbit bladder via urethra, then the
Firstly, the process of intravesical of floating hydrogel into rabbit bladder was detected by thermo-sensitive hydrogel solutioninjection was injected into bladder using catheter. After that, the hydrogel Firstly, the process of intravesical injection of floating hydrogel into rabbit bladder was detected and6). floated in bladder More information about floating process canbe seen ultrasoundformed (Figure Catheter wasimmediately. inserted into rabbit bladder viathe urethra, then the thermo-sensitive by ultrasound (Figure 6). Catheter was The inserted into image rabbit non-floating bladder via urethra, then the in the Video S1 injected (Supplementary Materials). hydrogel in rabbit hydrogel solution was into bladder using ultrasound catheter. After of that, the hydrogel formed and floated thermo-sensitive solution was injected into bladder catheter. After was that,unable the hydrogel bladder was hydrogel also captured (Figure S3). From the image, the using non-floating hydrogel to in bladder immediately. More information about the floating process canbe seen in the Video S1 formed and inattached bladdertoimmediately. More information about the floating process canbe seen float in floated urine and the bladder wall. (Supplementary Materials). The ultrasound image of non-floating hydrogel in rabbit in bladder in the Video S1 (Supplementary Materials). The ultrasound image of non-floating hydrogel rabbit was also captured (Figure S3). From the image, the non-floating hydrogel was unable to float in urine bladder was also captured (Figure S3). From the image, the non-floating hydrogel was unable to and attached bladder wall. to the bladder wall. float to in the urine and attached
Figure 6. Process of intravesically injection of floating hydrogel into rabbit bladder (detected by ultrasound). The white dashed curves in the pictures represented the rabbit bladder wall. The white dashed circles represented gels in rabbit bladder. The catheters are indicated with white arrows.
Then, we measured RhB release rate from non-floating and floating hydrogels, compared with Figure 6. Process of intravesically injection of floating hydrogel into rabbit bladder (detected by
in vivo 7). The first voiding of was triggered filling the rabbit bladder 2~10 Figurefree 6. RhB Process of(Figure intravesically injection ofurine floating hydrogelbyinto rabbit bladder (detected by ultrasound). The white dashed curves in the pictures represented the rabbit bladder wall. The white min after intravesical instillation of saline, free RhB solution, non-floating hydrogel or floating ultrasound). The white dashed curves in the pictures represented the rabbit bladder wall. The white dashed circles represented gels in rabbit bladder. Theofcatheters are indicated with white arrows. hydrogel (Figure S4). After intravesical instillation RhB solution, RhB concentration in bladder dashed circles represented gels in rabbit bladder. The catheters are indicated with white arrows. reached the peak immediately, but dropped sharply after the first voiding of urine. As to the
Then, we measured release rate non-floating and hydrogels, compared non-floating hydrogelRhB group, due to thefrom incapacity of floating in floating urine, it attached on the bladder with free RhB in vivo (Figure 7). The first voiding of urine was triggered by filling the rabbit bladder Then, we measured RhB release rate from non-floating and floating hydrogels, compared2~10 with free min after intravesical instillation of saline, free RhB solution, non-floating hydrogel or floating RhB in vivo (Figure 7). The first voiding of urine was triggered by filling the rabbit bladder 2~10 min hydrogel (Figure S4). After intravesical instillation of RhB solution, RhB concentration in bladder after intravesical instillation of saline, free RhB solution, non-floating hydrogel or floating hydrogel reached the peak immediately, but dropped sharply after the first voiding of urine. As to the non-floating hydrogel group, due to the incapacity of floating in urine, it attached on the bladder
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(Figure S4). After intravesical instillation of RhB solution, RhB concentration in bladder reached the peak immediately, but dropped sharply after the first voiding of urine. As to the non-floating hydrogel group, due to the incapacity of floating in urine, it attached on the bladder wall, so it 9caused severe Materials 2016, 9, 1005 of 14 bladder irritation. Consequently, most gels were expelled from bladder during the first voiding of so it caused severe bladder irritation. Consequently, most gels were expelled from bladder urine, andwall, only little hydrogel remained. In contrast, the intravesical delivery of floating hydrogel during the first voiding of urine, and only little hydrogel remained. In contrast, the intravesical showed sustained of RhB from gel. The concentration of RhB from floating hydrogel delivery2016, ofrelease floating hydrogel showed sustained release of RhB from gel.released The concentration of9 RhB Materials 9, 1005 of 14 reached 35.6% before the second voiding. hydrogel releasing drug, released from floating hydrogel reachedAfter 35.6%voiding, before thefloating second voiding. Afterremained voiding, floating wall, so itremained caused reached severe bladder most gels werebefore expelled from voiding, bladder hydrogel releasing drug,irritation. and RhB Consequently, concentration reached 23.2% the third and RhB concentration 23.2% before the third voiding, and 12.5% before the last voiding. during the first voiding of urine, andCompared only littletohydrogel remained. In contrast, the intravesical and 12.5% before the last voiding. free RhB and non-floating hydrogel, floating Compared to free RhB and non-floating hydrogel, floating hydrogel could significantly extend the delivery floating hydrogel extend showedthe sustained release gel. The concentration of RhB hydrogelofcould significantly residence time of ofRhB RhBfrom in bladder and hence increase the residence released time offrom RhBfloating in bladder andreached hence 35.6% increase the hydrogel before thebioavailability. second voiding. After voiding, floating bioavailability. hydrogel remained releasing drug, and RhB concentration reached 23.2% before the third voiding, and 12.5% before the last voiding. Compared to free RhB and non-floating hydrogel, floating hydrogel could significantly extend the residence time of RhB in bladder and hence increase the bioavailability.
Figure 7. Drug release of floating hydrogel, non-floating hydrogel and free RhB solution in vivo
Figure 7. (Mean Drug± release SD, n = 3).of floating hydrogel, non-floating hydrogel and free RhB solution in vivo (Mean ± SD, n = 3). Finally, the residual amount of RhB in rabbit bladder of normal saline, free RhB solution, RhB-loaded hydrogel RhB-loaded floating hydrogel groups were investigated Figure 7.non-floating Drug release of floating and hydrogel, non-floating hydrogel and free RhB solution in vivo Finally, the8).residual amount of RhB in rabbit bladder of normal saline, free RhB solution, (Figure (Mean ±The SD,results n = 3). showed that the fluorescence intensity of floating hydrogel group was much RhB-loaded non-floating and RhB-loaded floating hydrogel groups higher than free RhBhydrogel solution and non-floating hydrogel groups. No fluorescence of RhBwere could investigated be Finally, residual amount offluorescence RhB in residual rabbit bladder saline, free RhBtissue solution, in the theshowed normal saline group. The amountofofnormal in the hydrogel bladder waswas much (Figure 8).observed The results that the intensity ofRhB floating group RhB-loaded non-floating hydrogel and so RhB-loaded floating hydrogel were investigated proportional to the fluorescent the residual amount of RhB ingroups rabbit bladder of floating higher than free RhB solution andintensity, non-floating hydrogel groups. No fluorescence of RhB could (Figure 8). group The results that thethan fluorescence intensity of floating group was much hydrogel was showed much higher other groups. Besides, therehydrogel is no targeted delivery be observed in the normal saline group. The residual amount of RhB in the bladder higher than on freethe RhB solution and non-floating hydrogel No fluorescence RhB could betissue was mechanism floating hydrogel drug delivery systemgroups. selectively to the cancer of cells rather than proportional to thein fluorescent intensity, soThe the residual of drug RhB inbladder rabbit bladder observed the normal saline residual amount of RhB in the was the healthy cells. Therefore, thegroup. floating hydrogel was aamount promising carrier for tissue raising theof floating proportional to the fluorescent intensity, the residual amountthere of RhBisinno rabbit bladderdelivery of floatingmechanism efficiency of intravesical administration. hydrogel group was much higher than othersogroups. Besides, targeted hydrogel group was much higher than other groups. Besides, there is no targeted delivery on the floating hydrogel drug delivery system selectively to the cancer cells rather than the healthy mechanism on the floating hydrogel drug delivery system selectively to the cancer cells rather than cells. Therefore, the floating hydrogel washydrogel a promising drug carrier for raising the the efficiency of the healthy cells. Therefore, the floating was a promising drug carrier for raising intravesical administration. efficiency of intravesical administration.
Figure 8. Phase and fluorescence picture of frozen sections of bladder tissue to evaluate residual of RhB in the bladder. (a) Bright-field of frozen sections of bladder tissue; (b) Fluorescence images of frozen sections of bladder tissue, the fluorescence intensity of floating hydrogel group was much higher than free RhB solution and non-floating hydrogel groups. Figure 8. Phase and fluorescence picture of frozen sections of bladder tissue to evaluate residual of
Figure 8. Phase fluorescence pictureofoffrozen frozen sections of bladder to evaluate residual of RhB RhB in and the bladder. (a) Bright-field sections of bladder tissue; tissue (b) Fluorescence images of frozen (a) sections of bladderof tissue, the sections fluorescence floating(b) hydrogel group was much of frozen in the bladder. Bright-field frozen of intensity bladderoftissue; Fluorescence images than tissue, free RhBthe solution and non-floating hydrogel groups. hydrogel group was much higher than sections ofhigher bladder fluorescence intensity of floating free RhB solution and non-floating hydrogel groups.
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3.5. The Efficacy of Floating Hydrogel in Acute Bladder Injury Model 3.5. The Efficacy of Floating Hydrogel in Acute Bladder Injury Model Interstitial cystitis (IC) patients have typical symptoms such as pain, frequency, urgency and Interstitial (IC) to patients have typical symptoms such of asthe pain, frequency, urgency and nocturia, owing cystitis to damages the glycosaminoglycan (GAG) layer bladder [13]. Acute bladder nocturia, owing to damages to the glycosaminoglycan (GAG) layer interstitial of the bladder Acute injury model induced by protamine sulfate was commonly used to study cystitis.[13]. Protamine bladder injury model induced by protamine sulfate was commonly used to study interstitial cystitis. sulfate destroyed the GAG layer of bladder mucosa, thus increasing the permeability of bladder Protamine sulfate destroyed the GAG layerbeofattenuated bladder mucosa, thus increasing the permeability of mucosa. The protamine sulfate effect might by exogenous sulfated polysaccharide, such bladder mucosa. The protamine sulfate effect might be attenuated by exogenous sulfated as heparin. polysaccharide, such as We investigated theheparin. efficacy of saline, floating hydrogel, heparin solution and heparin-loaded We investigated theinefficacy of saline, floating hydrogel, solution heparin-loaded floating hydrogel groups preventing the permeation of RhB inheparin protamine sulfateand pretreated bladders. floating hydrogel groups in preventing the permeation of RhB in protamine sulfate pretreated The higher permeability of RhB indicated poorer efficacy of drugs. In our experiment, the permeability bladders. higherwas permeability RhB poorer efficacy of drugs. In our experiment, of bladderThe mucosa evaluatedofby theindicated fluorescence depth of RhB in bladder wall (Figurethe 9). permeability of bladder mucosa was evaluated by the fluorescence depth of RhB in bladder wall The fluorescence depth was indicated by the width of fluorescence zone in the bladder wall, and (Figure 9). The fluorescence was indicated the width of fluorescence in the bladder the fluorescence distributiondepth with width was alsoby plotted (Figure 9b,c). Figure zone 9d showed that the wall, and the fluorescence distribution with width was also plotted (Figure 9b,c). Figure 9d than showed fluorescence depth of RhB in the PS + saline group and PS + Gel group were much larger the that the fluorescence depth of RhB in the PS + saline group and PS + Gel group were much larger other two groups (p < 0.05), proving the capacity of heparin to reverse the wall leakage that PS caused. than the other two+groups (psolution < 0.05), proving the +capacity of heparingel to group reverseshowed the wallmuch leakage that Compared to PS heparin group, PS heparin-loaded poorer PS caused. Compared to PS + heparin solution group, PS + heparin-loaded gel group showed much permeation of RhB (p < 0.05), with width similar to control group, indicating better efficiency of poorer permeation of RhB 0.05),voiding with width towere control group, better efficiency heparin. Because the time(pof< first of allsimilar groups 2~10 min,indicating and the heparin solution of heparin. Because the time of first voiding of all groups were 2~10 min, and the heparin solution was eliminated during that time. However, floating hydrogel could stay in bladder and continued was eliminated during thatimproving time. However, floating hydrogelTherefore, could staythe in bladder continued to to release heparin, largely the efficacy of heparin. floating and hydrogel system release heparin, largely improving the efficacy of heparin. Therefore, the floating hydrogel system could successfully extend the residence time of drug and hence enhance drug efficacy. could successfully extend the residence time of drug and hence enhance drug efficacy.
Figure 9. The efficacy of different treatments in acute bladder injury model: (a) phase picture of Figure 9. The efficacy of different treatments in acute bladder injury model: (a) phase picture of frozen frozen sections of bladder tissue; (b) fluorescence picture of frozen sections of bladder tissue; (c) sections of bladder tissue; (b) fluorescence picture of frozen sections of bladder tissue; (c) fluorescence fluorescence intensity–distance profiles of different bladder frozen sections; and (d) penetration intensity–distance profiles of different bladder frozen sections; and (d) penetration depth of different depth of different bladder frozen sections. (Mean ± SD, n = 3). bladder frozen sections. (Mean ± SD, n = 3).
4. Discussion 4. Discussion The safety problem of medical material is a tough obstacle lying in its application from bench to The safety problem of medical material is a tough obstacle lying in its application from bench bedside. In this study, we chose Poloxamer 407 as the only ingredient of our delivery system. The to bedside. In this study, we chose Poloxamer 407 as the only ingredient of our delivery system. safety of P407 hydrogel in vivo has been widely studied by previous reports [14]. In terms of The safety of P407 hydrogel in vivo has been widely studied by previous reports [14]. In terms of biocompatibility studies in vivo, there was no damage observed in muscle cells after injection of 20% P407 [15]. Moreover, after intravesical instillation, the concentration of P407 in urine was much lower than 20%. Furthermore, preparation of this system was just shaking the P407 aqueous solution
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biocompatibility studies in vivo, there was no damage observed in muscle cells after injection of 20% P407 [15]. Moreover, after intravesical instillation, the concentration of P407 in urine was much lower than 20%. Furthermore, preparation of this system was just shaking the P407 aqueous solution for 20 s, which is extremely simple. Therefore, there is no safety problem in the clinical application of our system. The efficacy of our system is inspiring according to in vivo drug release and efficacy experiments, much better than non-floating hydrogels and drug solutions. P407 is polyoxyethylene-polyoxypropylene-polyoxyethylene (PEOn-PPOn-PEOn) tri-block copolymer, non-ionic surfactants, possessing biocompatible, thermo-sensitive, low toxicity, foamability and weak immunogenic properties [14,16]. In this study, its foamability enabled P407 to act as micro-bubbles source of the floating hydrogel system. By shaking the P407 solution, air was incorporated in the solution, a bubble rising in a surfactant solution accumulates surfactant molecules on its surface (the surfactant adsorption at the air/water interface) [17]. The number or growth rate of micro-bubbles increased with the concentration of surfactant (P407) in solution, as well as air amount incorporated in the solution (i.e., more shaking times) [18]. The stability of micro-bubbles in solution was positively correlated with the concentration or viscosity of surfactant [19]. Thus, micro-bubbles would suspend in solution for a period because of the viscosity of the P407 solution. When P407 solution converted to gel state (at 37 ◦ C), so the air micro-bubbles in gel could not move any more thus the micro-bubbles were “locked” in gel. When the gel changed to the solution state (at 0 ◦ C), the air micro-bubbles in solution would gradually dissipate away because of the stochastic nature of bubble coalescence in viscous liquids. We also have tested the gelation properties of the solution at 25 ◦ C, the gelation temperature of the hydrogel solution was about 11.5 ◦ C, so the hydrogel converted to the gel state from the solution state at 25 ◦ C. Since in clinical operation, the injection will be operated under room temperature, so we stored the gel solution at 0 ◦ C condition before injected to the bladder, the viscosity of 45% P407 was low (about 200 mPa·s) at this temperature. When take the gel solution under room temperature from 0 ◦ C condition, the gelation time of the gel solution was about 5 min after shaking quickly. Therefore, it has enough time to inject the gel solution into bladder. As the shear force have impact on the stability of the micro-bubbles during injection, we injected the hydrogel solution (3 mL) into the bladder quickly (about 10 s). The short injection time may reduce the impact of shear force. The floating state of hydrogel was confirmed by ultrasound after injection. We also used homogenizer to produce micro-bubbles. Hydrogel solution stirred for 10 s at the speed of 5000 rpm could float in water stably (Figure S5). Considering the convenience in clinical application, we used shaking by hand to replace machine. The time to float of hydrogel should be as short as possible since the short time can decrease the risk of urinary obstruction or bladder irritation. In this floating hydrogel system, the hydrogel could float immediately due to the whole hydrogel was full of micro-bubbles at the beginning by shaking the P407 solution. However, the time to float of hydrogels we previously developed were several minutes, because chemical reaction took time [7,8]. P407 formed micelles to entrap drugs, so drugs was released throughout the diffusion process of gel matrix. Drugs released with the process of P407 dissolution and ended once P407 dissolved completely [20]. The erosion time of hydrogel should be longer to extend the release time of drug. Increasing concentration and hydrogel volume of P407 will lengthen its erosion time and hence extend the release time of drug. Moreover, the viscosity of gels also increased with the concentration of the P407 [20]. Therefore, 45% P407 was chosen to obtain optimal erosion time and viscosity. The results showed that the erosion time of this floating hydrogel system (about 6 h/5 mL) was much longer than our previous floating hydrogel containing NH4 HCO3 (about 3 h/5 mL) [8]. These are due to the chemical reaction inside hydrogel accelerated the gel erosion. In this study, the drug release time in vivo was not consistent with that in vitro. The erosion time of the 3 mL floating hydrogel was about 3.5~4 h in vitro. However, in vivo release study showed that drug was released for about 10 h. The time discrepancy of drug release might result from the volume difference between bladder (5~40 mL) and beaker (200 mL). Another reason was the urine residues in
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the bladder [21]. In addition, the autofluorescence of rabbit urine might increase the measurement of RhB release amount. Moreover, the contraction of bladder during injection and gelation process will reduce the amount of micro-bubble generated and thus result in the reduction in erosion time in vivo. Bladder irritation is often induced by urinary infection or foreign bodies in the bladder (such as bladder stone or blood clots) [22]. In our experiments, most non-floating hydrogels were expelled from rabbit bladder after intravesical instillation. It indicated that non-floating hydrogel caused severe bladder irritation as it attached to the bladder wall, and caused the rabbit bladder detrusor forcefully contracted to expel it out. Compared to the non-floating hydrogel, the floating hydrogel was floating in urine and was not directly in contact with the bladder wall, so the floating hydrogel could avoid cause the bladder irritation. Previous reports have shown that patients of interstitial cystitis endure damage of the glycosaminoglycan (GAG) layer of urothelium [23]. The surface GAG layer has been proposed as a protective barrier that coats the transitional cell surface. Parsons has demonstrated that the increasing bladder mucosal permeability to solu-urea in patients with interstitial cystitis could be imitated by intravesical administration of protamine sulfate [24]. The protamine effect appeared to be reversed by treatments with exogenous sulfated polysaccharide (i.e., intravesical administration of heparin, hyaluronic acid, or pentosan polysulfate) [25]. Heparin was initially reported to relieve the symptoms of IC in the 1960s, and subsequent studies also proved its efficacy [26]. Its exact mechanism of action was not understood at that time. One hypothesis was that heparin provided exogenous sulfated polysaccharide which coated the epithelium to maintain the bladder mucosa integrity. In this study, rabbit acute bladder injury model was constructed by intravesical administration of protamine sulfate, and the efficacy of heparin loaded floating hydrogel was evaluated by fluorescence depth in the bladder wall. Our results showed that the heparin-loaded floating hydrogel was more effective than free heparin solution in alleviating the damage effects of protamine sulfate. Because the floating hydrogel released RhB persistently and extended the residence time of heparin in bladder, while the free heparin solution was eliminated of bladder along with the first voiding of urine. Our results also showed that the P407 hydrogel did not affect the bladder mucosal permeability to RhB. As the heparin served as the exogenous sulfated polysaccharide, the prolonged drug resident time in bladder increased the efficiency of heparin in the protection of bladder mucosal permeability. The floating hydrogel system was a promising drug delivery system in the treatment of IC patients whose bladder GAGs layer was damaged. 5. Conclusions This paper presents a new self-generating micro-bubbles floating hydrogel drug delivery system for intravesical instillation. The Matrix of floating hydrogel is P407 without any additives, addressing safety concerns for clinical use. The floating property of floating hydrogel avoids urinary obstruction and bladder irritation that conventional hydrogels cause, and improves drug efficacy by prolonging the residence time. In other words, this new drug delivery system is safe, simply prepared and efficient for future clinical use. Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/9/12/1005/s1. Figure S1: Effects of shaking time on erosion time (a) and gelation temperature (b) in hydrogel, Figure S2: Effects of shaking time on micro-bubbles number in hydrogel, Figure S3: Non-floating hydrogel in rabbit bladder, Figure S4: The time of first voiding after intravesical instillation, Figure S5: Hydrogel solution was homogenized, then hydrogel floats stably in water, Figure S6: Volume change of hydrogel in respect to the density of micro-bubble, Figure S7: Effect of environmental factors (temperature (a), humidity (b), and atmosphere pressure (c)) and the shaking force (d) on micro-bubble generation, Video S1: Floating hydrogel in vitro and vivo. Acknowledgments: This study was supported by National Natural Science Foundation (No. 81202474, 81273464 and 81473146), Changzhou Special Project of Biotechnology and Biopharmacy (No. CE20105006). Science Bridges China-Changzhou Biotechnology and Pharmaceutical Technology Special Project (Grant Number: CE20105006). J. Wu is sponsored by Qing Lan Project. Author Contributions: Hongqian Guo, Jinhui Wu and Yiqiao Hu conceived and designed the experiments; Tingsheng Lin and Xiaozhi Zhao performed the experiments; Tingsheng Lin analyzed the data; Huibo Lian,
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Junlong Zhuang, Qing Zhang, Wei Chen, Wei Wang, Guangxiang Liu and Suhan Guo contributed reagents/materials/analysis tools; Tingsheng Lin and Yifang Zhang wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.
References 1. 2. 3. 4.
5.
6. 7.
8.
9.
10.
11.
12. 13. 14. 15. 16.
17. 18. 19.
GuhaSarkar, S.; Banerjee, R. Intravesical drug delivery: Challenges, current status, opportunities and novel strategies. J. Controll. Release: Off. J. Controll. Release Soc. 2010, 148, 147–159. [CrossRef] [PubMed] Gasion, J.P.; Cruz, J.F. Improving efficacy of intravesical chemotherapy. Eur. Urol. 2006, 50, 225–234. [CrossRef] [PubMed] Tyagi, P.; Li, Z.; Chancellor, M.; De Groat, W.C.; Yoshimura, N.; Huang, L. Sustained intravesical drug delivery using thermosensitive hydrogel. Pharm. Res. 2004, 21, 832–837. [CrossRef] [PubMed] Men, K.; Liu, W.; Li, L.; Duan, X.; Wang, P.; Gou, M.; Wei, X.; Gao, X.; Wang, B.; Du, Y.; Huang, M. Delivering instilled hydrophobic drug to the bladder by a cationic nanoparticle and thermo-sensitive hydrogel composite system. Nanoscale 2012, 4, 6425–6433. [CrossRef] [PubMed] Zhang, D.; Sun, P.; Li, P.; Xue, A.; Zhang, X.; Zhang, H.; Jin, X. A magnetic chitosan hydrogel for sustained and prolonged delivery of Bacillus Calmette-Guerin in the treatment of bladder cancer. Biomaterials 2013, 34, 10258–10266. [CrossRef] [PubMed] Collado, A.; Chechile, G.E.; Salvador, J.; Vicente, J. Early complications of endoscopic treatment for superficial bladder tumors. J. Urol. 2000, 164, 1529–1532. [CrossRef] Lin, T.; Wu, J.; Zhao, X.; Lian, H.; Yuan, A.; Tang, X.; Zhao, S.; Guo, H.; Hu, Y. In situ floating hydrogel for intravesical delivery of adriamycin without blocking urinary tract. J. Pharm. Sci. 2014, 103, 927–936. [CrossRef] [PubMed] Lin, T.; Zhang, Y.; Wu, J.; Zhao, X.; Lian, H.; Wang, W.; Guo, H.; Hu, Y. A Floating Hydrogel System Capable of Generating CO Bubbles to Diminish Urinary Obstruction after Intravesical Instillation. Pharm. Res. 2014, 31, 2655–2663. [CrossRef] [PubMed] Buckton, G.; Machiste, E.O. Differences between dynamic and equilibrium surface tension of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymer surfactants (poloxamers P407, P237, and P338) in aqueous solution. J. Pharm. Sci. 1997, 86, 163–166. [CrossRef] [PubMed] Golemanov, K.; Denkov, N.D.; Tcholakova, S.; Vethamuthu, M.; Lips, A. Surfactant mixtures for control of bubble surface mobility in foam studies. Langmuir ACS J. Surf. Colloids 2008, 24, 9956–9961. [CrossRef] [PubMed] Shelke, S.; Shahi, S.; Jadhav, K.; Dhamecha, D.; Tiwari, R.; Patil, H. Thermoreversible nanoethosomal gel for the intranasal delivery of Eletriptan hydrobromide. J. Mater. Sci. Mater. Med. 2016, 27, 1–13. [CrossRef] [PubMed] Costa, P.; Sousa Lobo, J.M. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 2001, 13, 123–133. [CrossRef] Parsons, C.L.; Mulholland, S.G. Successful Therapy of Interstitial Cystitis with Pentosanpolysulfate. J. Urol. 1987, 138, 513–516. [PubMed] Dumortier, G.; Grossiord, J.L.; Agnely, F.; Chaumeil, J.C. A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm. Res. 2006, 23, 2709–2728. [CrossRef] [PubMed] Ricci, E.J.; Lunardi, L.O.; Nanclares, D.M.; Marchetti, J.M. Sustained release of lidocaine from Poloxamer 407 gels. Int. J. Pharm. 2005, 288, 235–244. [CrossRef] [PubMed] Shelke, S.; Shahi, S.; Jalalpure, S.; Dhamecha, D. Poloxamer 407-based intranasal thermoreversible gel of zolmitriptan-loaded nanoethosomes: Formulation, optimization, evaluation and permeation studies. J. Liposome Res. 2016, 26, 313–323. [CrossRef] [PubMed] Fdhila, R.B.; Duineveld, P.C. The effect of surfactant on the rise of a spherical bubble at high Reynolds and Peclet numbers. Phys. Fluids 1996, 8, 310–321. [CrossRef] Hetsroni, G.; Mosyak, A.; Pogrebnyak, E.; Sher, I.; Segal, Z. Bubble growth in saturated pool boiling in water and surfactant solution. Int. J. Multiphas Flow 2006, 32, 159–182. [CrossRef] Giribabu, K.; Ghosh, P. Binary coalescence of air bubbles in viscous liquids in presence of non-ionic surfactant. Can. J. Chem. Eng. 2008, 86, 643–650. [CrossRef]
Materials 2016, 9, 1005
20.
21. 22. 23. 24.
25. 26.
14 of 14
Zhang, L.; Parsons, D.L.; Navarre, C.; Kompella, U.B. Development and in vitro evaluation of sustained release poloxamer 407 (P407) gel formulations of ceftiofur. J. Controll. Release: Off. J. Controll. Release Soc. 2002, 85, 73–81. [CrossRef] Bruskewitz, R.C.; Iversen, P.; Madsen, P.O. Value of postvoid residual urine determination in evaluation of prostatism. Urology 1982, 20, 602–604. [CrossRef] Eckford, S.D.; Persad, R.A.; Brewster, S.F.; Gingell, J.C. Intravesical foreign bodies: Five-year review. Br. J. Urol. 1992, 69, 41–45. [CrossRef] [PubMed] Chancellor, M.B.; Yoshimura, N. Treatment of interstitial cystitis. Urology 2004, 63, 85–92. [CrossRef] [PubMed] Nickel, J.C.; Downey, J.; Morales, A.; Emerson, L.; Clark, J. Relative efficacy of various exogenous glycosaminoglycans in providing a bladder surface permeability barrier. J. Urol. 1998, 160, 612–614. [CrossRef] Parsons, C.L. The role of the urinary epithelium in the pathogenesis of interstitial cystitis/ prostatitis/urethritis. Urology 2007, 69, 9–16. [CrossRef] [PubMed] Parsons, C.L. Epithelial coating techniques in the treatment of interstitial cystitis. Urology 1997, 49, 100–104. [CrossRef] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
