BACKGROUND
cancer is a major public health problem in the world, which causes millions of death each year.
approximately  <dig>  million new cancer cases and more than  <dig>  deaths from cancer are projected to occur in  <dig> in usa  <cit> .
now, one in four deaths in usa is due to cancer.
as conventional therapy for cancer, chemotherapy has wide applications in clinical, which has been proven to be effective.
although chemotherapeutic agents may prolong the survival time of the patients, unfortunately most of them have severe side toxic effects, which would decline the life quality of patients.
most conventional methods for delivering chemotherapeutic agents, such as intravenous injection or oral ingestion, fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels.
novel controlled drug delivery systems  are designed to deliver drugs at predetermined rates for predefined periods at the target organ, which could be used to overcome the shortcomings of conventional drug formulations, therefore could diminish the side effects and improve the life quality of the patients  <cit> .
thus, a suitable controlled drug delivery system is extremely important for chemotherapy.
hydrogels are a special class of macromolecules, which could absorb much water while maintaining their integrity in water.
over the past decades, the stimuli-sensitive hydrogel has attracted increasing attention owing to their responsiveness to the environmental stimulus, including chemical substances and changes in temperature, ph, or electric field  <cit> .
the biodegradable thermosensitive physical crosslinked hydrogels have been extensively studied due to their great biodegradability, biocompatibility, and responsiveness to temperature.
therefore, biodegradable thermosensitive hydrogels have been investigated as in situ gel-forming system, such as controlled drug delivery, tissue repair, and cell encapsulation  <cit> .
poly-poly-poly triblock copolymer , known as pluronic or poloxamer, has been extensively studied as a potential drug delivery vehicle due to their excellent biocompatibility and thermosensitivity  <cit> .
these copolymers have been widely used as emulsifiers, wetting agents, and solubilizers  <cit> .
however, due to weak hydrophobicity of ppg block, the pluronic f <dig> copolymer forms a fast-eroding gel and could persist a few hours in vivo at most, which greatly restricted its application as in situ gel-forming controlled drug delivery system.
in our previous study, we prepared a new kind of biodegradable and injectable thermosensitive poly-poly-poly  hydrogel controlled drug delivery system  <cit> .
at low temperature, pece hydrogel is injectable flowing sol, which could be easily mixed with pharmaceutical agent, and it forms non-flowing gel at body temperature as sustained drug delivery site in vivo.
pcl and peg are biocompatible and have been widely used in several fda approved products  <cit> .
pcl is lack of toxicity and has great permeability  <cit> .
due to combination of great advantages of peg and pcl, the pece hydrogel might have great potential application in biomedical field.
in our last work  <cit> , we prepared a new biodegradable and injectable composite hydrogel based on pece and pluronic f <dig> copolymer.
the composite hydrogel undergoes sol-gel-sol transition, which is free flowing sol at room temperature and becomes a non-flowing gel at body temperature.
our last work mainly focused on the synthesis, in vivo gel formation and degradation assay, and toxicity evaluation of the composite hydrogel.
it is well known that pluronic copolymer forms a fast-eroding hydrogel which could not persist longer than a few hours.
and pece hydrogel could persist  <dig> weeks in vivo  <cit> .
according to our results, by simply altering the composition of pece and pluronic f <dig> copolymers, the in vivo sustained time of the composite hydrogel could be controlled, which could meet the different requirements of in vivo sustained time, therefore the composite hydrogel is very useful for its potential application as injectable in situ gel-forming drug delivery system.
in the present study, cytotoxicity and in vitro drug release behavior of the composite hydrogel were studied in detail.
many factors affecting in vitro drug release behavior of the composite hydrogel were investigated, including different kinds of drugs, initial drug loading, concentration of composite hydrogel, and chemical composition of the composite hydrogel.
by altering the composition of composite hydrogel, sol-gel-sol transition behavior and in vitro drug release behavior of the prepared composite hydrogel could be controlled, which was of great importance for their further application as injectable in situ gel-forming drug release system.
methods
materials
poly methyl ether , ε-caprolactone , pluronic f <dig> , hexamethylene diisocyanate , stannous octoate  <dig>  sigma, usa), dulbecco's modified eagle's medium , 3-- <dig> -diphenyl tetrazolium bromide , bovine serum albumin  and vb <dig>  were used without further purification.
honokiol  were isolated and purified in our lab  <cit> .
all the materials used in this article were analytic reagent  grade and used as received.
preparation of composite hydrogel
pece copolymer were synthesized and purified as reported previously  <cit> .
briefly, peg-pcl diblock copolymers were prepared by ring opening polymerization of ε-cl initiated by mpeg using stannous octoate as catalyst; peg-pcl-peg triblock copolymers were synthesized by coupling peg-pcl diblock copolymers using hmdi as coupling agent  <cit> .
the just-obtained pece block copolymers were dissolved in dichloromethane, and then reprecipitated from the filtrate using excess cold petroleum ether.
then, the mixture was filtered and vacuum dried to constant weight at room temperature.
the purified copolymers were kept in air-tight bags before further use.
the obtained pece copolymer was characterized by ftir , 1h-nmr , and gpc .
the mn and peg/pcl ratio of pece triblock copolymer calculated from 1h-nmr spectra was  <dig> and 960/ <dig> respectively.
macromolecular weight and macromolecular weight distribution  of pece triblock copolymer determined by gpc were  <dig> and  <dig>  respectively  <cit> .
preparation scheme of composite hydrogel was described in fig.
<dig>  aqueous pece solutions were prepared by dissolving pece copolymers in deionized water at a designated temperature then cooled to 4°c.
then, different amounts of pluronic f <dig> were dissolved in icy cold deionized water to a transparent solution.
subsequently, the obtained two solutions were mixed together under mild agitation to obtain a homogeneous liquid solution.
the final solution contained a given concentration and composition of the two copolymers to form the different composite hydrogel samples.
the composite hydrogels prepared in this work were listed in table  <dig>
sol-gel-sol phase transition behavior study
sol-gel-sol phase transition diagrams of composite hydrogels were recorded using test tube-inverting method  <cit> .
the sol-gel-sol transition was visually observed by inverting the vials, and conditions of sol and gel were defined as "flowing" and "non-flowing" in one minute, respectively.
in this work, the volume of the composite hydrogel solution was kept to  <dig>  ml in total, regardless of the concentration.
after incubated in water bath at 4°c for  <dig> minutes, the hydrogel samples were slowly heated at a rate of  <dig> °c/min, from 4°c to the temperature when the sol states occurred again.
cytotoxicity assay of composite hydrogel
cytotoxicity evaluation of composite hydrogel  was performed by 3- - <dig> -diphenyl tetrazolium bromide  assay using hek <dig> cells, which were grown in dmem with 10% of fetal bovine serum  and were cultured in a humidified atmosphere containing 5% co <dig> at 37°c.
cell suspensions were distributed in a 96-well plate at a density of  <dig> ×  <dig> cells/well in dmem medium with 10% of fbs for  <dig> hours.
then, the medium were replaced by  <dig> μl of fresh dmem per well containing different amounts of composite hydrogel copolymers  from  <dig> μg/ml to  <dig> μg/ml, respectively.
after  <dig> hours, the cell cultures were washed with pbs solution and mtt assay was conducted.
untreated cells were taken as control with 100% viability.
the cell cytotoxicity of composite hydrogel was defined as the relative viability  which correlates with amount of liable cells compared with cell control.
preparation of honokiol micelles
honokiol  micelles were prepared and characterized in our previous work  <cit> .
briefly, certain amount of hk was dissolved into et ac to form hk solution.
then, the prepared hk-et ac solution was introduced into  <dig> ml of f <dig> aqueous solution at the concentration of 5%w/w under extreme stirring by t <dig> .
about  <dig> min later, oil in water  emulsion well formed.
then, et ac was evaporated in rotator evaporator  and the hk micelles were obtained.
the content of hk in the prepared hk micelles suspension was determined by high-performance liquid chromatography .
at last, the hk micelles slurry was lyophilized and the power was stored at 4°c before further use.
in vitro drug release behavior from the composite hydrogel
release behavior of hydrophilic small molecular drugs
vb <dig> was used as model to determine the release behavior of hydrophilic small molecular drug from composite hydrogel in vitro.
<dig> μl of vb <dig> loaded composite hydrogel  were placed into  <dig> ml-eppendorf  tubes and allowed to gel in an incubator at 37°c for  <dig> h. then, the gels were immersed in  <dig> ml of pbs  and were shaken at  <dig> rpm at 37°c.
at specific time intervals, all the release media were removed and replaced by fresh release media.
after centrifuged at  <dig> rpm for  <dig> min, the supernatant of the removed release media were collected and stored at -20°c until analysis.
the collected supernatants were detected on uv spectrophotometer at  <dig> nm to determine the concentration of vb <dig>  the accumulatively released vb <dig> was calculated according to the following equation  <cit> :
q = cnvt + vsΣcn- <dig>
where q was accumulatively released weight, and cn was the vb <dig> concentration at time t. vt was the volume of medium , and vs was the volume of solution removed from supernatant .
release behavior of hydrophobic small molecular drugs
freshly prepared hk micelles loaded composite hydrogel were used to assay in vitro release behavior of hydrophobic small molecular drugs.
in detail,  <dig> μl of prepared hk micelles loaded composite hydrogel  were transferred into  <dig> ml-ep tubes and allowed to gel in an incubator at 37°c for  <dig> h. then, the gels were immersed in  <dig> ml of pbs  and were shaken at  <dig> rpm at 37°c.
at specific time intervals, all the release media were removed and replaced by fresh release media.
after centrifuged at  <dig> rpm for  <dig> min, the supernatant of the removed release media were collected and stored at -20°c until analysis.
the concentration of hk was determined by hplc instrument .
solvent delivery system equipped with a column heater and a plus autosampler.
detection was taken on a waters  <dig> detector.
chromatographic separations were performed on a reversed phase c <dig> column .
and the column temperature was kept at 28°c.
acetonitrile/water  was used as eluent at a flow rate of  <dig> ml/min.
the standard curve equation is: h = 105000*x+ <dig>  and the correlation coefficient is  <dig> .
release behavior of hydrophilic macromolecular protein drugs
in vitro release behavior of bsa, which was used as the model protein or peptide drug, from bsa loaded composite hydrogel was studied in detail.
the procedure was similar to section  <dig> .
<dig>  except that the initial drug loading amount were  <dig> mg and  <dig> mg, respectively.
the amount of bsa present in the supernatant was determined by bicinchoninic acid  assay using bca™ protein assay kit .
the sds-polyacrylamide gel electrophoretic  analysis was used to assay the stability of bsa in the supernatant.
all the release study experiments were repeated three times.
all data are expressed as the mean ± s.d.
scanning electron microscopy  of composite hydrogel
sem was employed to investigate morphology of composite hydrogel before and after drug release.
the composite hydrogels  were quickly frozen in liquid nitrogen and lyophilized at -45°c for  <dig> h. the composite hydrogels were sputtered with gold before observation.
in this study, morphology of prepared composite hydrogels was examined on jeol sem .
RESULTS
synthesis of pece copolymers
the synthesis of pece triblock copolymers has been reported in our previous work  <cit> .
briefly, ring-opening copolymerization of ε-cl onto mpeg was performed to synthesis peg-pcl diblock copolymers, and stannous octoate was used as catalyst.
peg-pcl diblock copolymers were then coupled using hmdi as coupling agent to produce the biodegradable peg-pcl-peg triblock copolymers.
temperature-dependent sol-gel-sol transition behavior
pece and pluronic f <dig> copolymers are both amphiphilic in nature, whose aqueous solution individually presented sol-gel-sol transition behavior.
the composite hydrogel prepared in this work were composed of the two copolymers.
as presented in table  <dig>  composite hydrogel based on pece and pluronic f <dig> copolymers from s <dig> to s <dig> all showed temperature-dependent reversible sol-gel-sol phase transition.
the composite hydrogel flowed freely at lower temperature, but became a non-flowing gel at body temperature about 37°c .
fig.
<dig> presented the sol-gel-sol phase transition diagrams of prepared composite hydrogel.
when the copolymer concentrations are above the critical gelation concentration , aqueous solutions of composite hydrogel changed from "sol" phase to "gel" phase with increase in temperature to the lower critical gelation temperature .
with further increase of temperature to upper critical gelation temperature , the sol phase occurs.
according to fig.
<dig>  pure pece hydrogel  and pure pluronic f <dig> hydrgel  both have a cgc of approximately  <dig> wt%, but s <dig> have a much wider gelation window than that of s <dig>  the ucgt of s <dig> at the concentration of  <dig> wt% and 35% was not detected from 0°c to 100°c.
the cgc of s <dig>  s <dig>  s <dig>  and s <dig> were  <dig> wt%,  <dig> wt%,  <dig> wt%, and  <dig> wt%, respectively, which were much higher than that of two pure hydrogels.
by mixing the two hydrogel together, the cgc of the composite hydrogel increased accordingly.
cgc of s <dig> increased approximately  <dig> wt% than that of s <dig>  whereas cgc of s <dig> increased approximately  <dig> wt% than that of s <dig>  this phenomenon indicated that concerning cgc, the influence of pluronic f <dig> hydrogel on pece hydrogel was more dramatic than that of pece hydrogel on pluronic f <dig> hydrogel.
as shown in fig.
<dig>  with increase in pece hydrogel content in composite hydrogel, the ucgt decreased significantly, whereas the lcgt increased slightly.
the ucgt of s <dig> hydrogel at concentration of  <dig> wt% and  <dig> wt% could not be detected from 0°c to 100°c, but the ucgt at concentration of  <dig> wt% and  <dig> wt% were detected in s <dig> and s <dig> hydrogel, respectively, due to increase in pece content.
therefore, it was obvious that sol-gel-sol transition behavior of composite hydrogel depended on the composition of the pece and pluronic f <dig> hydrogel.
in fact, by altering the composition of composite hydrogel, the temperature range of sol-gel-sol phase transition could be broadened to a certain extent, which might be very useful for their further application as injectable in situ gel-forming drug delivery system.
cytotoxicity study of composite hydrogel copolymers
the cytotoxicity of the prepared composite hydrogels  was evaluated by cell viability assay using hek  <dig> cells.
fig.
<dig> exhibited the hek  <dig> cell viability of composite hydrogel copolymer with different concentration gradient.
as shown in fig.
<dig>  with increase of composite hydrogel copolymer amount, hek  <dig> cell viability decreased accordingly.
however, the  <dig> cell viability of s <dig>  s <dig>  s <dig> and s <dig> copolymers were yet higher than  <dig> %,  <dig> %,  <dig> % and  <dig> %, respectively, even when the input copolymers were  <dig> μg per well.
according to fig.
<dig>  the hek  <dig> cell viability of composite hydrogel copolymer decreased as increase of composite hydrogel copolymers.
compared with s <dig> and s <dig> copolymers group, s <dig> copolymer has higher cell viability at  <dig> μg/well or lower, but cell viability decreased significantly at  <dig> μg/well or higher.
cell viability study implied that the composite hydrogel copolymers prepared in this paper were biocompatible with low cell cytotoxicity.
therefore, composite hydrogel could be regarded as safe drug delivery carrier and is very promising for in situ gel-forming controlled drug delivery system.
in vitro drug release profile of composite hydrogel
vb <dig>  hk, and bsa were used as model drugs to investigate the release behavior of hydrophilic small molecular drug, hydrophobic small molecular drug, and protein drug from drug loaded composite hydrogels, respectively.
effect of the hydrogel composition, initial drug loading, and concentration of composite hydrogel on in vitro drug release behavior of the composite hydrogel were investigated in detail, which were discussed as follows.
release behavior of hydrophilic small molecular drug
in vitro release profile of vb <dig> from composite hydrogel in pbs was studied, and the results were shown in fig.
<dig>  according to fig.
<dig>  vb <dig> could be released in a sustained period.
the hydrogel composition had great effect on vb <dig> release profile, and the results were shown in fig.
5–a.
s <dig> hydrogel disappeared completely in  <dig> hours with a cumulative release rate of approximately  <dig> %, whereas s <dig> and s <dig> hydrogel could maintain their integrity in the whole release period.
vb <dig> released faster and reached higher cumulative release rate  from s <dig> hydrogel compared to s <dig> hydrogel , which should be contributed to high composition  of fast-eroding pluronic f <dig> in s <dig> hydrogel.
effect of initial drug loading amount on release profile of s <dig> hydrogel was investigated.
as shown in fig.
5–b, s <dig> hydrogel containing twice amount of vb <dig> result in a significant decrease of cumulative release rate from  <dig> % to  <dig> %, which was constant to our previous work.
with the same initial drug loading amount but lower hydrogel concentration  of s <dig>  vb <dig> released faster and reached higher cumulative release rate  compared to  <dig> wt% concentration s <dig> hydrogel .
due to the higher composition of fast-eroding pluronic f <dig> copolymer and lower hydrogel concentration, the  <dig> wt% s <dig> hydrogel was completely eroded in  <dig> hours.
in  <dig> wt% hydrogel, an initial burst release of  <dig> % of loaded vb <dig> occurred in the first one hour, followed by release of  <dig> % in two days, whereas, in  <dig> wt% hydrogel, the cumulative release rate of one hour, two days and  <dig> days were  <dig> %,  <dig> %, and  <dig> %, respectively.
release behavior of hydrophobic small molecular drug
hk, as multi-functional drug, have great potential application in human disease therapy, especially in cancer therapy.
previously, a rapid separation approach to isolate and purify hk had been developed using high-capacity high-speed counter-current chromatography  by chen et al in our lab  <cit> .
for its great potential application and high hydrophobicity, hk was chosen for the hydrophobic model drug in this in vitro drug release study.
due to high hydrophobicity, hk could not be well-disperse in the composite hydrogel to form homogeneous solution.
hk micelles were employed to solve above-mentioned problem.
the obtained hk micelles with average particle size of  <dig>  nm and polydisperse index  of  <dig>  could be well-dispersed in water, and it was stable.
only pluronic f <dig>  a composition of composite hydrogel, was remained in the hk micelles, which would not affect in vitro release behavior of composite hydrogel.
the release behavior of hk from composite hydrogel was performed and the cumulative release profile was presented in fig.
<dig>  in fig.
6–a, with increase in content of pluronic f <dig> copolymer from 0%  to 100% , cumulative release rate and burst release rate  increased from  <dig> % to  <dig> % and from  <dig> % to  <dig> %, respectively.
according to fig.
6–b, with increase of initial drug loading amount, the cumulative release rate of hk decreased dramatically from  <dig> % to  <dig> % in a 14-day period.
as shown in fig.
6–c, lower concentration of composite hydrogel led to higher cumulative release rate in a shorter time.
compared with vb12release profile, cumulative release rate and burst release rate of hk were much lower, which should be contributed to the high hydrophobicity of hk.
release behavior of hydrophilic macromolecular protein drugs
in vitro release behavior of protein or peptide model drug from composite hydrogel was investigated, and the data were summarized in fig.
7–a, b, and 7c.
bsa could be released slowly from composite hydrogel in an extended period.
as presented in fig.
7–a, b, and 7c, effects of hydrogel composition, initial drug loading amount, and hydrogel concentration on bsa release profile were investigated in detail.
the results were similar to the influence of these factors on hk release profile.
sds-page was performed to evaluate the stability of bsa in the in vitro release period.
according to fig.
7–d, the major band for bsa appeared at about  <dig> kd  according to the protein marker, which showed that bsa was stable in the experimental period.
thus, composition of composite hydrogel, initial drug loading amount, and hydrogel concentration substantially affected the drug release behavior of composite hydrogel, where higher pluronic f <dig> content, lower initial drug loading amount, or lower hydrogel concentration resulted in higher cumulative release rate, which means drug release rate of composite hydrogel could be controlled by simply altering the composition of pece and pluronic f <dig> copolymers.
it is obvious that the influence of above-mentioned three factors is more dramatic on hk and bsa than vb <dig>  due to the great water solubility, vb <dig> was released very fast from the composite hydrogel, which could weaken the influence of the factors.
besides the factors mentioned above, physical and chemical property of the drugs played an important role in their release behavior.
according to fig.
<dig> to fig.
<dig>  compared with hydrophobic small molecular drug, hydrophilic small molecular drug reached a higher cumulative release rate in a shorter period.
in addition, cumulative release rate of hydrophilic small molecular drug was much higher than that of hydrophilic large molecular drugs.
morphology of composite hydrogel
interior morphology of composite hydrogel before and  <dig> hours after drug released was investigated by sem.
the composite hydrogels were frozen in liquid nitrogen and lyophilized for  <dig> h before the test.
according to fig.
<dig>  all the hydrogel samples showed porous three-dimension structure, but the shape and mesh size of pores in the hydrogel were different.
as shown in fig.
8–a, s <dig> hydrogel before drug release presented approximately spherical pore with small mesh size.
s <dig> hydrogel , composed of 60% pece hydrogel and 40% pluronic f <dig> hydrogel, also showed spherical pores, but have larger mesh size compared to s <dig> hydrogel.
the morphology of s <dig> and s <dig> hydrogel suggested that the composition of composite hydrogel have great influence on their interior structure, which dramatically affected the drug release behavior of composite hydrogel.
eight hours after drug released, s <dig> hydrogel could maintain its integrity, but the hydrogel surface eroded .
in fig.
8–d, s <dig> hydrogel after immersed in pbs for  <dig> hours showed large pores and cracks, due to the fast-eroding of pluronic f <dig> from the composite hydrogel.
CONCLUSIONS
a series of novel biodegradable and thermosensitive composite hydrogel were successfully prepared in this work.
the obtained composite hydrogel underwent sol-gel-sol transition with increase in temperature, which was flowing sol at ambient temperature and became non-flowing gel at body temperature.
by varying the composition of pece and pluronic f <dig> copolymers, sol-gel-sol transition behavior and in vitro drug release profile of composite hydrogel could be adjusted, which was very useful for its potential applications as in situ gel-forming controlled drug delivery system.
abbreviations
pece: poly-poly-poly copolymer; peg: poly; pcl: poly; hplc: high-performance liquid chromatography; dds: controlled drug delivery systems; vb12: vitamin b12; hk: honokiol,; bsa: bovine serum albumin; mpeg: poly methyl ether; f127: pluronic f <dig>  hmdi: hexamethylene diisocyanate); sn2: stannous octoate; dmem: dulbecco's modified eagle's medium; mtt: 3-- <dig> -diphenyl tetrazolium bromide.
competing interests
the authors declare that they have no competing interests.
authors' contributions
qzy, wyq, and gcy designed the experiments.
and the research funds were supported by qzy and wyq.
gcy carried out experiments, analyzed the data, and wrote the manuscript; qzy corrected the manuscript.
ss and yjl participated in the mtt cytotoxicity study of the hydrogels.
zxl participated in the in vitro release study from the composite hydrogels.
dpw, gg and fsz participated in synthesizing hydrogel and analyzing the data.
all authors approved and read the final manuscript.

 cancer is a major public health problem in the world, which causes millions of death each year. approximately million new cancer cases and more than deaths from cancer are projected to occur in in usa. now, one in four deaths in usa is due to cancer. as conventional therapy for cancer, chemotherapy has wide applications in clinical, which has been proven to be effective. although chemotherapeutic agents may prolong the survival time of the patients, unfortunately most of them have severe side toxic effects, which would decline the life quality of patients. most conventional methods for delivering chemotherapeutic agents, such as intravenous injection or oral ingestion, fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels. novel controlled drug delivery systems are designed to deliver drugs at predetermined rates for predefined periods at the target organ, which could be used to overcome the shortcomings of conventional drug formulations, therefore could diminish the side effects and improve the life quality of the patients. thus, a suitable controlled drug delivery system is extremely important for chemotherapy. hydrogels are a special class of macromolecules, which could absorb much water while maintaining their integrity in water. over the past decades, the stimuli-sensitive hydrogel has attracted increasing attention owing to their responsiveness to the environmental stimulus, including chemical substances and changes in temperature, ph, or electric field. the biodegradable thermosensitive physical crosslinked hydrogels have been extensively studied due to their great biodegradability, biocompatibility, and responsiveness to temperature. therefore, biodegradable thermosensitive hydrogels have been investigated as in situ gel-forming system, such as controlled drug delivery, tissue repair, and cell encapsulation. poly-poly-poly triblock copolymer, known as pluronic or poloxamer, has been extensively studied as a potential drug delivery vehicle due to their excellent biocompatibility and thermosensitivity. these copolymers have been widely used as emulsifiers, wetting agents, and solubilizers. however, due to weak hydrophobicity of ppg block, the pluronic f copolymer forms a fast-eroding gel and could persist a few hours in vivo at most, which greatly restricted its application as in situ gel-forming controlled drug delivery system. in our previous study, we prepared a new kind of biodegradable and injectable thermosensitive poly-poly-poly hydrogel controlled drug delivery system. at low temperature, pece hydrogel is injectable flowing sol, which could be easily mixed with pharmaceutical agent, and it forms non-flowing gel at body temperature as sustained drug delivery site in vivo. pcl and peg are biocompatible and have been widely used in several fda approved products. pcl is lack of toxicity and has great permeability. due to combination of great advantages of peg and pcl, the pece hydrogel might have great potential application in biomedical field. in our last work, we prepared a new biodegradable and injectable composite hydrogel based on pece and pluronic f copolymer. the composite hydrogel undergoes sol-gel-sol transition, which is free flowing sol at room temperature and becomes a non-flowing gel at body temperature. our last work mainly focused on the synthesis, in vivo gel formation and degradation assay, and toxicity evaluation of the composite hydrogel. it is well known that pluronic copolymer forms a fast-eroding hydrogel which could not persist longer than a few hours. and pece hydrogel could persist weeks in vivo. according to our results, by simply altering the composition of pece and pluronic f copolymers, the in vivo sustained time of the composite hydrogel could be controlled, which could meet the different requirements of in vivo sustained time, therefore the composite hydrogel is very useful for its potential application as injectable in situ gel-forming drug delivery system. in the present study, cytotoxicity and in vitro drug release behavior of the composite hydrogel were studied in detail. many factors affecting in vitro drug release behavior of the composite hydrogel were investigated, including different kinds of drugs, initial drug loading, concentration of composite hydrogel, and chemical composition of the composite hydrogel. by altering the composition of composite hydrogel, sol-gel-sol transition behavior and in vitro drug release behavior of the prepared composite hydrogel could be controlled, which was of great importance for their further application as injectable in situ gel-forming drug release system. methods materials poly methyl ether, ε-caprolactone, pluronic f, hexamethylene diisocyanate, stannous octoate sigma, usa), dulbecco's modified eagle's medium, 3-- -diphenyl tetrazolium bromide, bovine serum albumin and vb were used without further purification. honokiol were isolated and purified in our lab. all the materials used in this article were analytic reagent grade and used as received. preparation of composite hydrogel pece copolymer were synthesized and purified as reported previously. briefly, peg-pcl diblock copolymers were prepared by ring opening polymerization of ε-cl initiated by mpeg using stannous octoate as catalyst; peg-pcl-peg triblock copolymers were synthesized by coupling peg-pcl diblock copolymers using hmdi as coupling agent. the just-obtained pece block copolymers were dissolved in dichloromethane, and then reprecipitated from the filtrate using excess cold petroleum ether. then, the mixture was filtered and vacuum dried to constant weight at room temperature. the purified copolymers were kept in air-tight bags before further use. the obtained pece copolymer was characterized by ftir, 1h-nmr, and gpc. the mn and peg/pcl ratio of pece triblock copolymer calculated from 1h-nmr spectra was and 960/ respectively. macromolecular weight and macromolecular weight distribution of pece triblock copolymer determined by gpc were and respectively. preparation scheme of composite hydrogel was described in fig. aqueous pece solutions were prepared by dissolving pece copolymers in deionized water at a designated temperature then cooled to 4°c. then, different amounts of pluronic f were dissolved in icy cold deionized water to a transparent solution. subsequently, the obtained two solutions were mixed together under mild agitation to obtain a homogeneous liquid solution. the final solution contained a given concentration and composition of the two copolymers to form the different composite hydrogel samples. the composite hydrogels prepared in this work were listed in table sol-gel-sol phase transition behavior study sol-gel-sol phase transition diagrams of composite hydrogels were recorded using test tube-inverting method. the sol-gel-sol transition was visually observed by inverting the vials, and conditions of sol and gel were defined as "flowing" and "non-flowing" in one minute, respectively. in this work, the volume of the composite hydrogel solution was kept to ml in total, regardless of the concentration. after incubated in water bath at 4°c for minutes, the hydrogel samples were slowly heated at a rate of °c/min, from 4°c to the temperature when the sol states occurred again. cytotoxicity assay of composite hydrogel cytotoxicity evaluation of composite hydrogel was performed by 3- - -diphenyl tetrazolium bromide assay using hek cells, which were grown in dmem with 10% of fetal bovine serum and were cultured in a humidified atmosphere containing 5% co at 37°c. cell suspensions were distributed in a 96-well plate at a density of × cells/well in dmem medium with 10% of fbs for hours. then, the medium were replaced by μl of fresh dmem per well containing different amounts of composite hydrogel copolymers from μg/ml to μg/ml, respectively. after hours, the cell cultures were washed with pbs solution and mtt assay was conducted. untreated cells were taken as control with 100% viability. the cell cytotoxicity of composite hydrogel was defined as the relative viability which correlates with amount of liable cells compared with cell control. preparation of honokiol micelles honokiol micelles were prepared and characterized in our previous work. briefly, certain amount of hk was dissolved into et ac to form hk solution. then, the prepared hk-et ac solution was introduced into ml of f aqueous solution at the concentration of 5%w/w under extreme stirring by t. about min later, oil in water emulsion well formed. then, et ac was evaporated in rotator evaporator and the hk micelles were obtained. the content of hk in the prepared hk micelles suspension was determined by high-performance liquid chromatography. at last, the hk micelles slurry was lyophilized and the power was stored at 4°c before further use. in vitro drug release behavior from the composite hydrogel release behavior of hydrophilic small molecular drugs vb was used as model to determine the release behavior of hydrophilic small molecular drug from composite hydrogel in vitro. μl of vb loaded composite hydrogel were placed into ml-eppendorf tubes and allowed to gel in an incubator at 37°c for h. then, the gels were immersed in ml of pbs and were shaken at rpm at 37°c. at specific time intervals, all the release media were removed and replaced by fresh release media. after centrifuged at rpm for min, the supernatant of the removed release media were collected and stored at -20°c until analysis. the collected supernatants were detected on uv spectrophotometer at nm to determine the concentration of vb the accumulatively released vb was calculated according to the following equation: q = cnvt + vsΣcn- where q was accumulatively released weight, and cn was the vb concentration at time t. vt was the volume of medium, and vs was the volume of solution removed from supernatant. release behavior of hydrophobic small molecular drugs freshly prepared hk micelles loaded composite hydrogel were used to assay in vitro release behavior of hydrophobic small molecular drugs. in detail, μl of prepared hk micelles loaded composite hydrogel were transferred into ml-ep tubes and allowed to gel in an incubator at 37°c for h. then, the gels were immersed in ml of pbs and were shaken at rpm at 37°c. at specific time intervals, all the release media were removed and replaced by fresh release media. after centrifuged at rpm for min, the supernatant of the removed release media were collected and stored at -20°c until analysis. the concentration of hk was determined by hplc instrument. solvent delivery system equipped with a column heater and a plus autosampler. detection was taken on a waters detector. chromatographic separations were performed on a reversed phase c column. and the column temperature was kept at 28°c. acetonitrile/water was used as eluent at a flow rate of ml/min. the standard curve equation is: h = 105000*x+ and the correlation coefficient is. release behavior of hydrophilic macromolecular protein drugs in vitro release behavior of bsa, which was used as the model protein or peptide drug, from bsa loaded composite hydrogel was studied in detail. the procedure was similar to section. except that the initial drug loading amount were mg and mg, respectively. the amount of bsa present in the supernatant was determined by bicinchoninic acid assay using bca™ protein assay kit. the sds-polyacrylamide gel electrophoretic analysis was used to assay the stability of bsa in the supernatant. all the release study experiments were repeated three times. all data are expressed as the mean ± s.d. scanning electron microscopy of composite hydrogel sem was employed to investigate morphology of composite hydrogel before and after drug release. the composite hydrogels were quickly frozen in liquid nitrogen and lyophilized at -45°c for h. the composite hydrogels were sputtered with gold before observation. in this study, morphology of prepared composite hydrogels was examined on jeol sem.  synthesis of pece copolymers the synthesis of pece triblock copolymers has been reported in our previous work. briefly, ring-opening copolymerization of ε-cl onto mpeg was performed to synthesis peg-pcl diblock copolymers, and stannous octoate was used as catalyst. peg-pcl diblock copolymers were then coupled using hmdi as coupling agent to produce the biodegradable peg-pcl-peg triblock copolymers. temperature-dependent sol-gel-sol transition behavior pece and pluronic f copolymers are both amphiphilic in nature, whose aqueous solution individually presented sol-gel-sol transition behavior. the composite hydrogel prepared in this work were composed of the two copolymers. as presented in table composite hydrogel based on pece and pluronic f copolymers from s to s all showed temperature-dependent reversible sol-gel-sol phase transition. the composite hydrogel flowed freely at lower temperature, but became a non-flowing gel at body temperature about 37°c. fig. presented the sol-gel-sol phase transition diagrams of prepared composite hydrogel. when the copolymer concentrations are above the critical gelation concentration, aqueous solutions of composite hydrogel changed from "sol" phase to "gel" phase with increase in temperature to the lower critical gelation temperature. with further increase of temperature to upper critical gelation temperature, the sol phase occurs. according to fig. pure pece hydrogel and pure pluronic f hydrgel both have a cgc of approximately wt%, but s have a much wider gelation window than that of s the ucgt of s at the concentration of wt% and 35% was not detected from 0°c to 100°c. the cgc of s s s and s were wt%, wt%, wt%, and wt%, respectively, which were much higher than that of two pure hydrogels. by mixing the two hydrogel together, the cgc of the composite hydrogel increased accordingly. cgc of s increased approximately wt% than that of s whereas cgc of s increased approximately wt% than that of s this phenomenon indicated that concerning cgc, the influence of pluronic f hydrogel on pece hydrogel was more dramatic than that of pece hydrogel on pluronic f hydrogel. as shown in fig. with increase in pece hydrogel content in composite hydrogel, the ucgt decreased significantly, whereas the lcgt increased slightly. the ucgt of s hydrogel at concentration of wt% and wt% could not be detected from 0°c to 100°c, but the ucgt at concentration of wt% and wt% were detected in s and s hydrogel, respectively, due to increase in pece content. therefore, it was obvious that sol-gel-sol transition behavior of composite hydrogel depended on the composition of the pece and pluronic f hydrogel. in fact, by altering the composition of composite hydrogel, the temperature range of sol-gel-sol phase transition could be broadened to a certain extent, which might be very useful for their further application as injectable in situ gel-forming drug delivery system. cytotoxicity study of composite hydrogel copolymers the cytotoxicity of the prepared composite hydrogels was evaluated by cell viability assay using hek cells. fig. exhibited the hek cell viability of composite hydrogel copolymer with different concentration gradient. as shown in fig. with increase of composite hydrogel copolymer amount, hek cell viability decreased accordingly. however, the cell viability of s s s and s copolymers were yet higher than %, %, % and %, respectively, even when the input copolymers were μg per well. according to fig. the hek cell viability of composite hydrogel copolymer decreased as increase of composite hydrogel copolymers. compared with s and s copolymers group, s copolymer has higher cell viability at μg/well or lower, but cell viability decreased significantly at μg/well or higher. cell viability study implied that the composite hydrogel copolymers prepared in this paper were biocompatible with low cell cytotoxicity. therefore, composite hydrogel could be regarded as safe drug delivery carrier and is very promising for in situ gel-forming controlled drug delivery system. in vitro drug release profile of composite hydrogel vb hk, and bsa were used as model drugs to investigate the release behavior of hydrophilic small molecular drug, hydrophobic small molecular drug, and protein drug from drug loaded composite hydrogels, respectively. effect of the hydrogel composition, initial drug loading, and concentration of composite hydrogel on in vitro drug release behavior of the composite hydrogel were investigated in detail, which were discussed as follows. release behavior of hydrophilic small molecular drug in vitro release profile of vb from composite hydrogel in pbs was studied, and the results were shown in fig. according to fig. vb could be released in a sustained period. the hydrogel composition had great effect on vb release profile, and the results were shown in fig. 5–a. s hydrogel disappeared completely in hours with a cumulative release rate of approximately %, whereas s and s hydrogel could maintain their integrity in the whole release period. vb released faster and reached higher cumulative release rate from s hydrogel compared to s hydrogel, which should be contributed to high composition of fast-eroding pluronic f in s hydrogel. effect of initial drug loading amount on release profile of s hydrogel was investigated. as shown in fig. 5–b, s hydrogel containing twice amount of vb result in a significant decrease of cumulative release rate from % to %, which was constant to our previous work. with the same initial drug loading amount but lower hydrogel concentration of s vb released faster and reached higher cumulative release rate compared to wt% concentration s hydrogel. due to the higher composition of fast-eroding pluronic f copolymer and lower hydrogel concentration, the wt% s hydrogel was completely eroded in hours. in wt% hydrogel, an initial burst release of % of loaded vb occurred in the first one hour, followed by release of % in two days, whereas, in wt% hydrogel, the cumulative release rate of one hour, two days and days were %, %, and %, respectively. release behavior of hydrophobic small molecular drug hk, as multi-functional drug, have great potential application in human disease therapy, especially in cancer therapy. previously, a rapid separation approach to isolate and purify hk had been developed using high-capacity high-speed counter-current chromatography by chen et al in our lab. for its great potential application and high hydrophobicity, hk was chosen for the hydrophobic model drug in this in vitro drug release study. due to high hydrophobicity, hk could not be well-disperse in the composite hydrogel to form homogeneous solution. hk micelles were employed to solve above-mentioned problem. the obtained hk micelles with average particle size of nm and polydisperse index of could be well-dispersed in water, and it was stable. only pluronic f a composition of composite hydrogel, was remained in the hk micelles, which would not affect in vitro release behavior of composite hydrogel. the release behavior of hk from composite hydrogel was performed and the cumulative release profile was presented in fig. in fig. 6–a, with increase in content of pluronic f copolymer from 0% to 100%, cumulative release rate and burst release rate increased from % to % and from % to %, respectively. according to fig. 6–b, with increase of initial drug loading amount, the cumulative release rate of hk decreased dramatically from % to % in a 14-day period. as shown in fig. 6–c, lower concentration of composite hydrogel led to higher cumulative release rate in a shorter time. compared with vb12release profile, cumulative release rate and burst release rate of hk were much lower, which should be contributed to the high hydrophobicity of hk. release behavior of hydrophilic macromolecular protein drugs in vitro release behavior of protein or peptide model drug from composite hydrogel was investigated, and the data were summarized in fig. 7–a, b, and 7c. bsa could be released slowly from composite hydrogel in an extended period. as presented in fig. 7–a, b, and 7c, effects of hydrogel composition, initial drug loading amount, and hydrogel concentration on bsa release profile were investigated in detail. the results were similar to the influence of these factors on hk release profile. sds-page was performed to evaluate the stability of bsa in the in vitro release period. according to fig. 7–d, the major band for bsa appeared at about kd according to the protein marker, which showed that bsa was stable in the experimental period. thus, composition of composite hydrogel, initial drug loading amount, and hydrogel concentration substantially affected the drug release behavior of composite hydrogel, where higher pluronic f content, lower initial drug loading amount, or lower hydrogel concentration resulted in higher cumulative release rate, which means drug release rate of composite hydrogel could be controlled by simply altering the composition of pece and pluronic f copolymers. it is obvious that the influence of above-mentioned three factors is more dramatic on hk and bsa than vb due to the great water solubility, vb was released very fast from the composite hydrogel, which could weaken the influence of the factors. besides the factors mentioned above, physical and chemical property of the drugs played an important role in their release behavior. according to fig. to fig. compared with hydrophobic small molecular drug, hydrophilic small molecular drug reached a higher cumulative release rate in a shorter period. in addition, cumulative release rate of hydrophilic small molecular drug was much higher than that of hydrophilic large molecular drugs. morphology of composite hydrogel interior morphology of composite hydrogel before and hours after drug released was investigated by sem. the composite hydrogels were frozen in liquid nitrogen and lyophilized for h before the test. according to fig. all the hydrogel samples showed porous three-dimension structure, but the shape and mesh size of pores in the hydrogel were different. as shown in fig. 8–a, s hydrogel before drug release presented approximately spherical pore with small mesh size. s hydrogel, composed of 60% pece hydrogel and 40% pluronic f hydrogel, also showed spherical pores, but have larger mesh size compared to s hydrogel. the morphology of s and s hydrogel suggested that the composition of composite hydrogel have great influence on their interior structure, which dramatically affected the drug release behavior of composite hydrogel. eight hours after drug released, s hydrogel could maintain its integrity, but the hydrogel surface eroded. in fig. 8–d, s hydrogel after immersed in pbs for hours showed large pores and cracks, due to the fast-eroding of pluronic f from the composite hydrogel.  a series of novel biodegradable and thermosensitive composite hydrogel were successfully prepared in this work. the obtained composite hydrogel underwent sol-gel-sol transition with increase in temperature, which was flowing sol at ambient temperature and became non-flowing gel at body temperature. by varying the composition of pece and pluronic f copolymers, sol-gel-sol transition behavior and in vitro drug release profile of composite hydrogel could be adjusted, which was very useful for its potential applications as in situ gel-forming controlled drug delivery system. abbreviations pece: poly-poly-poly copolymer; peg: poly; pcl: poly; hplc: high-performance liquid chromatography; dds: controlled drug delivery systems; vb12: vitamin b12; hk: honokiol,; bsa: bovine serum albumin; mpeg: poly methyl ether; f127: pluronic f hmdi: hexamethylene diisocyanate); sn2: stannous octoate; dmem: dulbecco's modified eagle's medium; mtt: 3-- -diphenyl tetrazolium bromide. competing interests the authors declare that they have no competing interests. authors' contributions qzy, wyq, and gcy designed the experiments. and the research funds were supported by qzy and wyq. gcy carried out experiments, analyzed the data, and wrote the manuscript; qzy corrected the manuscript. ss and yjl participated in the mtt cytotoxicity study of the hydrogels. zxl participated in the in vitro release study from the composite hydrogels. dpw, gg and fsz participated in synthesizing hydrogel and analyzing the data. all authors approved and read the final manuscript. 
vitamin b, honokiol, and bovine serum albumin were used as model drugs to investigate the in vitro release behavior of hydrophilic small molecular drug, hydrophobic small molecular drug, and protein drug from the composite hydrogel respectively. chemical composition of composite hydrogel, initial drug loading, and hydrogel concentration substantially affected the drug release behavior. by varying the composition, sol-gel-sol transition and in vitro drug release behavior of the composite hydrogel could be adjusted. and it was flowing sol at ambient temperature but became non-flowing gel at body temperature. cytotoxicity of the composite hydrogel was conducted by cell viability assay using human hek cells. most conventional methods for delivering chemotherapeutic agents fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels. the higher pluronic f content, lower initial drug loading amount, or lower hydrogel concentration resulted in higher cumulative release rate. the cell viability of composite hydrogel copolymers were yet higher than %, even when the input copolymers were μg per well. the results showed that composite hydrogel prepared in this paper were biocompatible with low cell cytotoxicity, and the drugs in this work could be released slowly from composite hydrogel in an extended period, which suggested that the composite hydrogel might have great potential applications in biomedical fields. novel controlled drug delivery systems are designed to deliver drugs at predetermined rates for predefined periods at the target organ and overcome the shortcomings of conventional drug formulations therefore could diminish the side effects and improve the life quality of the patients. thus, a suitable controlled drug delivery system is extremely important for chemotherapy. a novel biodegradable thermosensitive composite hydrogel, based on poly-poly-poly and pluronic f copolymer, was successfully prepared in this work, which underwent thermosensitive sol-gel-sol transition. all the above-mentioned drugs in this work could be released slowly from composite hydrogel in an extended period.
 most conventional methods for delivering chemotherapeutic agents fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels. novel controlled drug delivery systems are designed to deliver drugs at predetermined rates for predefined periods at the target organ and overcome the shortcomings of conventional drug formulations therefore could diminish the side effects and improve the life quality of the patients. thus, a suitable controlled drug delivery system is extremely important for chemotherapy.  a novel biodegradable thermosensitive composite hydrogel, based on poly-poly-poly and pluronic f copolymer, was successfully prepared in this work, which underwent thermosensitive sol-gel-sol transition. and it was flowing sol at ambient temperature but became non-flowing gel at body temperature. by varying the composition, sol-gel-sol transition and in vitro drug release behavior of the composite hydrogel could be adjusted. cytotoxicity of the composite hydrogel was conducted by cell viability assay using human hek cells. the cell viability of composite hydrogel copolymers were yet higher than %, even when the input copolymers were μg per well. vitamin b, honokiol, and bovine serum albumin were used as model drugs to investigate the in vitro release behavior of hydrophilic small molecular drug, hydrophobic small molecular drug, and protein drug from the composite hydrogel respectively. all the above-mentioned drugs in this work could be released slowly from composite hydrogel in an extended period. chemical composition of composite hydrogel, initial drug loading, and hydrogel concentration substantially affected the drug release behavior. the higher pluronic f content, lower initial drug loading amount, or lower hydrogel concentration resulted in higher cumulative release rate.  the results showed that composite hydrogel prepared in this paper were biocompatible with low cell cytotoxicity, and the drugs in this work could be released slowly from composite hydrogel in an extended period, which suggested that the composite hydrogel might have great potential applications in biomedical fields. 