| IntroductionMyocardial infarction（MI）,which is mainly induced by acute,persistent ischemia or hypoxia of cardiac tissues,is a major cause of mortality and disability worldwide.Pathologically,MI could lead to various pathological changes including oxidative stress,alteration in energy metabolism,apoptosis,release of inflammatory factors,and cardiomyocyte death,which in turn trigger the profibrotic and hypertrophic signaling cascades to develop into ventricular remodeling and even heart failure.Supramolecular peptide hydrogels,which are synthesized by peptide-based molecule assembly through non-covalent interactions and allow administration of local injection,show higher biocompatibility and biodegradation than polymer hydrogels.Thus,supramolecular peptide hydrogels are commonly used to encapsulate drugs or growth factors to treat related disease.Transient receptor potential vanilloid 1（TRPV1）is a nonselective cation-gated channel,which could be activated by a series of physical or chemical noxious stimuli such as capsaicin,heat stimulation,acidic environment,or endogenous mediators.Previous studies have shown that over-activation of TRPV1 plays an important role in the pathological changes post myocardial ischemia.Gross et al.derived an 11-amino acid peptide from C-terminus TRPV1 TRP domain（R701-S711）and conjugated it with TAT47-57 to yield V1-Cal（YGRKKRRQRRRGSGRAITILDTEKS）for its intracellular delivery.They found that V1-Cal could effectively reduce myocardial infarct size.Specifically,in isolated and in vivo myocardial ischemia-reperfusion（MIR）rodent models,V1-Cal efficiently reduced MI by limiting MIR injury with 61.2% and 60.7%,respectively.Since V1-Cal has a short half-life for conventional administration,a recent study reported that,by continuous pump delivery of V1-Cal,it reduced the injury of vascular endothelial cells and promoted the angiogenesis post arterial injury in diabetes rodent models.But to the best of our knowledge,using a biomaterial as a carrier for sustained release of V1-Cal to treat MI has not been reported.Based on above literature research,naturally we think,could we co-assemble V1-Cal with a supramolecular hydrogelator to form a hydrogel for sustained release of V1-Cal to reduce ventricular remodeling,improve cardiac function,and finally treat MI? To achieve this goal,we co-assembled V1-Cal with a well-known hydrogelator Nap-Phe-Phe-Tyr-OH（NapFFY）,and indeed obtained a new supramolecular hydrogel V1-Cal/NapFFY which may decrease infarct size and fibrosis,while improved cardiac function of the rat model.Part One.Synthesis,feasibility,reliability and safety of Gel V1-Cal/NapFFY Materials and methods1.Synthesis of hydrogelatorsCompounds of Phe-Phe-Tyr-OH（FFY）,Phe-Phe-Phe-Tyr-OH（FFFY）Nap-Phe-Phe-Tyr-OH（NapFFY）,Fmoc-Phe-Phe-Tyr-OH（FmocFFY）was synthesized by solid phase peptide synthesis（SPPS）and purified by HPLC.2.Critical aggregation concentrations（CACs）The hydrogelator of different concentration gradients were configured with PBS,and the p H was adjusted to 8.0 with fully stirred.Then,the fluorescence measurement method was used to analyze the intensity of emission wavelength by using 254 nm excitation wave of benzene ring or 268 nm excitation wave of Nap,and the Origin software was used to calculate the critical aggregation concentrations.3.Minimum gelation concentrations（MGCs）MGC was calculated with inverted-tube test.The hydrogelator of different concentrations added or not added with drugs were arranged in different small reagent bottles,and gelatinizing experiments were conducted according to the conditions explored in the early stage.The hydrogelator dissolved in PBS,adjusted p H to 8.0,then heated to 55 °C and cooled to room temperature.Turn the small reagent bottle upside down and observe whether the material in the reagent bottle will flow down,that is,whether it will be gelation.4.Synthetic route for 125I-V1-Cal.Injecting 100 μL V1-Cal in a closed flask,than 125I-Na I was added.Chloramine-T solution was added to start the iodination reaction.It was then shaken at room temperature.The total radioactivity of iodine-125 in hydrogel was calibrated by FCY type radiotomographic scanner and CRC-25 R Dose Calibrator.Results1.Hydrogelators screeningWe designed and synthesized four hydrogelators for carrying V1-Cal: NapFFY,FFY,FFFY and FmocFFY.According to the calculation of their CACs,MGCs and rheology,the performances of self-assembly and co-assembly with V1-Cal were analyzed,and the best hydrogelator was selected.It was found that the NapFFY hydrogelator had the best gelling performance and the ability to co-assemble with V1-Cal.2.Preparations of hydrogels Gel NapFFY,Gel V1-Cal/NapFFY,and Gel V1-Scr/NapFFYAfter synthesis and characterizations of NapFFY,we investigated its hydrogelation condition with（or w/o）V1-Cal according to previous methods.To obtain Gel NapFFY,we firstly dissolved 10 mg NapFFY powder in 1 m L phosphate buffered saline（PBS,0.01 M,p H 7.4）at 25 °C and adjusted the solution p H value to 8.0,then heated up the solution to 55 °C until it became clear.A transparent hydrogel at 1.0 wt% formed 30 min later after the solution cooling down to room temperature（25 °C）.For Gel V1-Cal/NapFFY hydrogel,10 mg NapFFY hydrogelator and 1 mg V1-Cal were dissolved in 1 m L PBS（0.01 M,p H 7.4）at 25 °C,then the gel was obtained with the same method as above.To prove the vital role of V1-Cal in the treatment of MI,here we scrambled the peptide sequence of V1-Cal to prepare another hydrogel Gel V1-Scr/NapFFY.3.CACs and MGCs of Gel NapFFY,Gel V1-Scr/NapFFY,and Gel V1-Cal/NapFFYTo further determine the gelation ability of NapFFY,V1-Cal/NapFFY and V1-Scr/NapFFY,we investigated their critical aggregation concentrations（CACs）and minimum gelation concentrations（MGCs）.Plots of fluorescence emission intensity versus concentration revealed two regimes,indicating critical aggregation concentration（CAC）of 16.6 μM for NapFFY,16.0 μM for V1-Cal/NapFFY,and 16.4 μM for V1-Scr/NapFFY,respectively.According to their CAC values,we calculated the co-assembly efficiency of V1-Cal with NapFFY and V1-Scr with NapFFY was 90.8%.The inverted tube test indicated that the CGCs for Gel NapFFY,Gel V1-Cal/NapFFY,and Gel V1-Scr/NapFFY were measured to be 0.25 ± 0.05 m M.The similar CAC and CGC values of these three hydrogels indicated that they have similar self-assembly capabilities.4.Rheology,CD spectra,and TEM characterizations of Gel NapFFY,Gel V1-Scr/NapFFY,and Gel V1-Cal/NapFFYNext we evaluated the viscoelastic properties of the obtained hydrogels（Gel NapFFY,Gel V1-Scr/NapFFY,and Gel V1-Cal/NapFFY）using rheology.Firstly we studied the dynamic strain scanning of the three hydrogels.All the storage modulus（G’）and loss modulus（G’’）values of the three hydrogels showed a weak dependence ranging from 0.01% to 1% of their strains（with G’ dominating G’’）at 1.0 wt%,indicating that all these studied samples are hydrogels.We conducted dynamic frequency scanning of these three hydrogels by setting the strain amplitude at 0.1%.At the strain amplitude of 1.0%,all G’ and G’’ values of the three hydrogels increased with the increase of frequency from 0.1 to 10 Hz.In addition,their G’ values were several times higher than those of G’’,indicating that all these hydrogels can tolerate external forces.Meanwhile,both G’ and G’’ values of Gel V1-Scr/NapFFY are close to those of Gel V1-Cal/NapFFY,respectively,indicating that Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY have close mechanical strengths.Interestingly,both Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY were elastically stronger than Gel NapFFY,suggesting that co-assembly between the peptide（V1-Scr or V1-Cal）with the hydrogelator NapFFY significantly enhances the mechanical strength of Gel NapFFY.After rheological tests,circular dichroism（CD）spectra of these three hydrogels were obtained to investigate the molecular packing of their corresponding hydrogelators.CD spectroscopy measurements indicated that Gel NapFFY exhibited a similar spectrum as Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY.In detail,CD spectrum of Gel NapFFY shows a remarkable positive Cotton effect at 202 nm and a clear negative CD absorption at 232 nm,indicating β-sheet secondary structures formed in the hydrogel.A remarkable positive Cotton effect at 202 nm and a clear negative CD absorption at 220 nm were also observed in CD spectrum of Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY.These results indicated that the encapsulation of V1-Cal or V1-Scr almost did not interfere with the molecular arrangements of the hydrogelators in the hydrogels.CD spectra of NapFFY also indicated that,when its concentration was lower than its CAC it did not form any secondary structure,while it formed above β-sheet secondary structure at concentrations higher than its CAC.Then we conducted transmission electron microscopy（TEM）observation to investigate the internal networks in these three hydrogels.In general,all the hydrogels were composed of long and flexible nanofibers.And very close nanofiber density between Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY was observed.However,the nanofiber densities of Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY were much higher than that of Gel NapFFY,which is consistent with above rheological results that Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY were mechanically stronger than Gel NapFFY.All these results indicated that the co-assembly between the peptide（V1-Scr or V1-Cal）and NapFFY does not affect the microscopic morphology of the formed hydrogels,but obviously enhances the mechanical strength of the related hydrogels.5.Cumulative release of V1-Cal or V1-Scr from Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY in vitro,respectivelyAfter rheology tests and TEM observations of the hydrogels,we studied in vitro release of V1-Cal or V1-Scr from Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY,respectively.Firstly,using HPLC analysis,we obtained the standard calibration curves of Vl-Cal and Vl-Scr by plotting the relationship between the peptide concentrations and its HPLC peak areas.After preparing Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY hydrogel as described above,we added 1 m L PBS（0.01 M,p H 7.4）to 200 μL Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY,and incubated it at 37 ℃.At different times（1,2,3,5,8,or 14 day）,1 m L of supernatant was collected for HPLC analysis.Then the culture mixture was replenished with 1 m L PBS immediately.V1-Cal and V1-Scr were released continuously within 14 days.In vitro release monitoring results showed that the cumulative release amount of V1-Cal and V1-Scr was 27.8 ± 8.4% and 24.4 ± 4.8% at day 3,respectively.The cumulative release amount of V1-Cal or V1-Scr increased with time and approached to 62.1 ± 8.2% or 60.8 ± 6.1% at day 14,respectively.The release rate of the peptide was high in the beginning 5 days and then gradually decreased with time.These experimental results indicated that both V1-Cal and V1-Scr were released from respective Gel V1-Cal/NapFFY and Gel V1-Scr/NapFFY at a sustainable manner,and suitable for in vivo experiments of MI treatment.6.Stability test of V1-Cal and V1-ScrThen we conducted the stability study of the peptides in PBS buffer（p H 7.4,10 m M）.HPLC analysis clearly indicated that either V1-Cal or V1-Scr was quite stable in PBS up to 16 days.7.Cytotoxicity evaluation of V1-Scr,V1-Cal,Gel NapFFY,Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFYThe viabilities of H9C2 cells treated with the hydrogels（Gel NapFFY,Gel V1-Scr/NapFFY,or Gel V1-Cal/NapFFY）were higher than those of cells treated with 0.1 mg/m L peptides（V1-Scr or V1-Cal）or non-treated（Control）.This suggests that the hydrogels with（or w/o）co-assembled peptides（V1-Scr or V1-Cal）could promote the proliferation of H9C2 cells.All these above results indicate that V1-Scr,V1-Cal,Gel NapFFY,Gel V1-Scr/NapFFY,or Gel V1-Cal/NapFFY is highly compatible to H9C2 cells and could be applied for following in vivo experiments.Part Two.Gel V1-cal /NapFFY on myocardial infarction in rats in vivo Materials and methods1.Myocardial infarction animal modelMyocardial infarction model was established by ligating the left anterior descending branch of rats.In Sham group,the coronary arteries were not ligated.The other groups were ligation of the anterior descending coronary artery.The peripheral area of myocardial infarction was injected with 5-point myocardial injection method.Hydrogel,V1-CAL or PBS were injected according to groups.20 μL per dot,100 μL in total.2.Terminal deoxynucleotidyl transferase mediated d UTP nick-end labelling（TUNEL）assayAccording to the instructions of manufacturer,apoptotic cells in the border zone of myocardial infarct 3 days after operation were analyzed by TUNEL staining using fluorescence labeled in situ cell death detection kit（Roche）.3.Western blotThe heart samples were incubated overnight at 4 °C with primary antibodies against GAPDH,TRPV1 and p-TRPV1 respectively.Samples were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37 °C.The immunoreactive proteins were visualized by ECL and autoradiography.4.Hematoxylin and eosin（H＆E）stainingOn day 28,major organs of mice were were fixed with 10% formalin over 48 h,embedded in paraffin,cut into 5 μm sections for H＆E staining,and evaluated using light microscopy.5.Immunohistochemistry stainingImmunohistochemistry was carried out to identify angiogenesis.Blood vessels were stained with a primary antibody against CD31（GB12063,Servicebio）（1:500）.Sections image was taken by fluorescence microscope（Zeiss,German）.The quantification of the angiogenesis was calculated using Image J software（NIH）.6.Enzyme-linked immunosorbent assay（ELISA）On day 1,3,7 after MI operation,blood samplings were obtained from tail vein of rats.TNF-alpha,Interleukin（IL）-6,IL-17 A levels in each serum sample were detected by using corresponding ELISA kits（ABclonal,China）.7.EchocardiographyTransthoracic echocardiography was performed at day 28 after MI with a Visual Sonics（VINNO 6 VET）equipped with a 18-MHz imaging transducer.M-mode tracings were recorded at the papillary muscle level to measure the left ventricular ejection fraction（EF）and fractional shortening（FS）,systolic left ventricular internal dimension（LVIDs）,diastolic left ventricular internal dimension（LVIDd）.8.Masson’s trichrome stainingThe rats were sacrificed and their hearts were harvested on day 28 after MI operation.Masson’s trichrome-staining was then performed for histological studies.Below data were obtained using Image J software（NIH）: the infarction area,the left ventricular wall thickness,and the fibrosis area in the border zone.Results1.Apoptosis,inflammatory factors,and TRPV1 expression analyses in MI ratsBoth V1-Cal and V1-Cal/NapFFY significantly alleviated cell apoptosis in the border area,with the latter more effective than the former.Quantitatively,the percentage of apoptotic cells was 3.8 ± 0.5% in MI group,but reduced to 2.1 ± 0.3% in V1-Cal group,or 0.8 ± 0.2% in V1-Cal/NapFFY group.Collectively,V1-Cal/NapFFY exhibited a stronger protective effect against apoptosis than V1-Cal.Compared with those in Sham group,expression levels of TNF-alpha and IL-6 in rats of all other five groups elevated markedly on day 1 and day 3 after MI,and the levels of IL-17 A in rats of other five groups raised to a high level on day 7 post-MI.Compared to those in MI,NapFFY,and V1-Scr/NapFFY groups,expression levels of all these three inflammatory factors in rats of V1-Cal and V1-Cal/NapFFY groups were obviously lower,indicating that the elevations of these inflammatory factors in MI were effectively inhibited by both V1-Cal or Gel V1-Cal/NapFFY treatment.And Gel V1-Cal/NapFFY treatment showed the best inhibitory effect on these inflammatory factors.Compared with those in Sham group,total TRPV1 level and p-TRPV1 level in the border region of rats in MI group increased by 3.6-fold and 3.1-fold,respectively.However,compared with those in MI group,total TRPV1 level and p-TRPV1 level were obviously lower in V1-Cal and V1-Cal/NapFFY groups,indicating that the elevated expressions of total TRPV1 and p-TRPV1 were suppressed by both V1-Cal and Gel V1-Cal/NapFFY.Again,Gel V1-Cal/NapFFY showed the strongest inhibition on both total TRPV1 and p-TRPV1 expressions among the four treated groups.2.Restoration of cardiac functionCompared with the rats in Sham group,MI rats showed significant ventricular dysfunction at the end of 4 weeks post MI,as indicated by the wall motion abnormalities from their echocardiography.But the ventricular dysfunction of the MI rats was largely recovered after Gel V1-Cal/NapFFY treatment.Echocardiography parameter measurements indicated that,compared with rats in Sham group,left ventricular ejection fraction（EF）and fractional shortening（FS）of MI rats significantly decreased,while their left ventricular internal diameter at end systole（LVIDs）and left ventricular internal diameter at end diastole（LVIDd）increased.Quantitative analysis showed that,four weeks after Gel V1-Cal/NapFFY treatment,EF value of the MI rats was improved from 48.9 ± 4.1% to 73.6 ± 3.3%.3.Promoting angiogenesisImmunohistostaining against CD31+,a typical mature endothelial marker,were conducted.Compared with those in Sham group,the percentage of CD31+ area of rats in MI group increased.Nevertheless,compared with that in MI group,the percentage of CD31+ area was obviously higher in V1-Cal or Gel V1-Cal/NapFFY group.Again,among the five treated groups,Gel V1-Cal/NapFFY group possessed the highest percentage of CD31+ area.4.Effects on ventricular remodelingIn general,massive fibrosis and ventricular remodeling were inhibited by Gel V1-Cal/NapFFY and V1-Cal treatments,but not by Gel NapFFY or Gel V1-Scr/NapFFY treatment.Specifically,Gel V1-Cal/NapFFY treatment reduced the infarct size from 41.0 ± 5.0% to 22.0 ± 2.4%,fibrosis area from 20.8 ± 3.3% to 5.9 ± 1.6%,while increased the wall thickness from 708.0 ± 121.2 to 1297.7 ± 185.4 μm of the MI rats.Again,V1-Cal also showed remodeling reduction effect on the MI rats but was less potent than Gel V1-Cal/NapFFY.Consistently,neither Gel NapFFY nor Gel V1-Scr/NapFFY treatment showed remodeling reduction effect on the MI rats.5.Evaluation of the sustainable release of V1-Cal from Gel V1-Cal/NapFFY in vivoFinally,we evaluated the in vivo release of V1-Cal from Gel V1-Cal/NapFFY using isotope labelling method.Firstly,in the presence of chloramine-T and Na125 I,the tyrosine（Y）in V1-Cal was labeled with 125 I.Then we used 125I-V1-Cal and NapFFY to prepare hydrogel according to above method.125 I labeled hydrogel was injected to 5 points along the infarct areas of one rat,and 125 I levels in heart,blood,liver,spleen,lung,and kidney were monitored at day 1 and 3 days post operation,so as to calculate the release and metabolism of V1-Cal in vivo.In vivo release monitoring results showed that the residual amount of 125I-V1-Cal in the hearts of rats was 50.7 ± 4.7% and 16.8 ± 5.4% at day 1 and day 3,respectively,maintained the highest concentration among all tissue and organs studied.The quantitative statistical results of the distribution of 125I-V1-Cal in blood and other tissues（liver,spleen,lung,kidney）at day 1 and day 3 day post injection.The results showed that,except heart,lung had the second high concentration of V1-Cal,followed by blood,kidney,spleen,and liver.Above experimental results proved that V1-Cal was slowly released from Gel V1-Cal/NapFFY at a sustainable manner in vivo.6.Biological safety in vivoTo further evaluate the systemic toxicity of our hydrogels,after treatment,we took the major organs（liver,spleen,lung,and kidney）from the rats for H＆E staining.Compared with the organs in control group,no pathological change was observed in the organs of these five experimental groups.All these results indicated that the hydrogels have high biological safety and are suitable for in vivo applications.ConclusionsIn conclusion,in order to more effectively reduce ventricular remodeling and improve cardiac function in a rat MI model,we co-assembled the therapeutic peptide V1-Cal with a well-studied hydrogelator NapFFY to prepare the supramolecular hydrogel Gel V1-Cal/NapFFY for sustained release of the peptide.Rheology and TEM tests of Gel V1-Cal/NapFFY showed that the co-assembly of V1-Cal with NapFFY increased mechanical strength of Gel NapFFY,enabling GelV1-Cal/NapFFY more suitable for myocardial injection to treat MI.Cumulative release profile of V1-Cal from Gel V1-Cal/NapFFY in vitro indicated that V1-Cal was released from Gel V1-Cal/NapFFY at a sustainable manner for more than two weeks.Cytotoxicity and proliferation results showed that Gel V1-Cal/NapFFY was non-toxic to but stimulated the proliferation of H9C2 cells.In vivo experiments indicated that,among all five experimental groups,Gel V1-Cal/NapFFY had the best effect on the decrease of TRPV1 expression and activation,apoptosis reduction and the release of inflammatory factors in a MI rat model.Moreover,Gel V1-Cal/NapFFY showed the best effect on cardiac function improvement,reverse effect on post-MI fibrosis and remodeling of the rats post MI.We envision that our Gel V1-Cal/NapFFY could be used to reduce ventricular remodeling and improve cardiac function post MI in clinic in the near future. |
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