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Radiatittn ptei^maHmof^rug cai-n»»s for aexamethasone and I tegafur based on polyCn-tsbprfljiyiafcrylanildel hydrogels
, ''°™»'5°X«M8.NgtiyenVanBWi,TianIfengDi<5;NguyeiiTHTlibnL Hoang Phiiong JBiao, Hiam DuyOuons Tran Mnh Quynh
BanoilrradtallanCenterlmATOM.No,S,MinhBai, TuUem.Hanoi (Received 26Se6niaiy 2015, accepted. 02A|)nl2015)
^ ^ P o I y ( N - i . p p i < ^ „ ^ t a n i d e ) (PM-AM). based hydn>gela ™a the lower edtical aolutiw l ^ m t o e (Lcsp near the human body-tempemture hav. be™, oblain«i ftom 10"/. solution, of N-
^ Z ^ ^ t ^ T ° 5 f ° "^""^^ '"'•^'^'^ " - " " " i * P « « 1 - ' °»the mitial ratio of Nffi
dri J ^ f 7 ^ '^ " ^ '"^''"^'' "^'^ ' y ^ e e k were about 48,6 and 95 7 ms ner . of
~ ; r " '^-^°'"--"»'^'-*j-- *-^-M- .^«™«w« .,,>, ,„,^, o^pdcitys
1. WmODUCXION , 1 , a „ ^ ^ ^ predetennined rates during the Hydrogel is a network of hydrephilio "^^"^ " ^ " " ^ °^''"'°- ™ ' otaracteristio has.
polymers that oan sweU in water and hold a ''°™ ™"' *• ""'"^"""^ * e dlsivbaefa of large amount of water while maintaining their '=°'n'""'°™l "tags [1-4], The hydrogels structure, hi practice, high swelling rate is an '=™'="™S ™li "sensor" properties can important property of hydrogels. In this case, a ^ ^'^° "^"^'^^' ™1™= phase transitions or fcee dimensional network is formed by ^ ' ' " ' ° ' '"'^^^ transition upon only mianre Shomical or physical crosslinking between the °'™«°^ ™ the environmental condition, polymer chainsunder the hydrogen boding, van ^''°'='"=. «>= stimuU-sensitive hydrogels ate fa- Waals mteractions, or physical "'''^'''^"''''''"^'"f "toveloping drug delivery entanglement, Hydrogels have been /"^"°^ ''^- ^^^ temperature-responsive intensively studied and applied for ' ' * ° ^ * ™ P t o ' ^ l y the most popular olass development of smart drug earners, where the " " ' '^'^ studied and applied as the dmg hydrogels can protect the dmg ftom hostile ^"^^^ Among that, poly(N- environments. They can also be apphed as "°P™Pyl'«^iylaniiile) (PNIPAAm) is the most carriers for controlled release dmgs by " " " i ' ^ *=™°-s=nsitive hydrogels with lower changing the gel stmcture in response to ™f ° ^ sohition temperature (LCST) of about environmental sthouli, and intentled to deUver ^^°''^' ^ ° ' ° * ""^ tenfljeiature, the hydrogel is
, , . . . „ y Society and Vietoam Alomic Energy Instimte
JL'VDIATION PREPARATION OF DRUG CARRIERS FOR DEXAMETHASONE AND TEGAFUR , hydrated and becomes hydrophilic. whereas it
would shnnk to become collapsed, dehydrated and hydrophobic form when temperature was above the LCST This is due to the breakdown of hydrophdie hydrophobic balance in the network structure [6]
Fortunately, the LCST can be changed by adjusting the ratio of the hydrophilic and hydrophobic segment of the polymer. The most popular way is to synthesize copolymers of hydrophobic (PNIPAAm) and hydrophilic (DMA) monomers with LCST closed to the human body temperature [7-9]
Several techniques have been reported on lhe synthesis of the hydrogels The crosslinking network can be formed during radical polymerization or copolymerization of monomers, which act as crosslinking agents.
While the chemical polymerization usually requires the presence of additives, which may contaminate the products Those initiators and catalysts do not require m case of radiation-mduced polymerization During irradiation, radicals produce on the polymer chains by the homolytic scission, such as C-H bonds, even C-C backbones. On the other hand, radiolysis of water molecules generates hydroxyl radicals which ean attack polymer chains also resulting in the formation of macro-radicals and then covalent bonds wilhin crosslinked siructure [10]
PNIPAAm can be synthesized from NIPA by radiation polymerization in both solid state and solution [10] fn aqueous solution, PNIPAAm can easily absorb water 10 become hydrogels And the properties of obtained PNIPAM hydrogels can be modified by adjusting monomer concentrations and irradiation conditions [11], Copolymerization of NIPA and other monomers results in the copolymers with more versatile properties, such as faster responsibility when heating and
thermo-sensitive hydrogels with additional stimuli- In our previous studies, some PNIPAAm based hydrogels have been prepared in aqueous solution by gamma irradiation [8,12]
To be glucocorticosteroid with anti- inflammatory and immuno-suppressant effecis, dexamethasone (DEX) lias been recenlly applied to treat some cancer, but its conventionai prolonged administration usually causes the undesired side effects [13]
Tegafur is a chemotherapeutic fluorouracil prodrug used m
the trealment of cancers that can transfer lo 5- fluorouracil during metabolism. The ma|or side effects of tegafur are similar to fluorouracil and mciude myelo-suppression, central neurotoxicity and ga strc mtest inal toxicity, which is the dosc-limiting side effect nf tegafur. Therefore, the use of these prodrugs should be controlled in order lo reduce iheir side effects
In this study, poiy{PNlPAAm-co- DMA) copolymerhydrogelswere oblained '' from the N I P A / D M A solutions by gamma irradiation at 20 kGy as indicated in our previous studies [7]. The swelling degrees of the resulting hydrogclswere investigaledwilh N I P A / D M A ratios in distilled water at room temperature Their loading capacities and release behaviors ofdexamethasonc and tcgafuras were determined in distilled water and simulated body fluid using UV-Vis spectroscopy,The influence of the solution temperature on the release rale of the products was also investigated with time al 2 5 , 37 and 40"C under constant shaking rate, fn vitro skin irritation tesl for these drug hydrogels were determined with OECD (TG 404) at National Institute of Drug Quality Conlrol
"T
HOANG DANG SANG et aL , n . EXPEKIMENTS
* A. Materials
N-isn)rpybciylamide 98% (NIPA), NJI- tiimothylaciylamide (DMA) monomers were purchased fiom Wako Pore Chemical b e , Japan, Nitrogen gas at industrial-giade purity was obtained ftom DucGiang Chemical
">mpany, Vietnam. Other chemicals for preparation of artificial body fluid were bought flom Merck, Getmany, Dexamethasone sodium phosphate and legafiir, chemotherapy prodrugs used m anti.cancer treatments were obtained fiom the national institute of drug quality conlrol. All chemicals were used as received.
B. Sample preparatton and irradiatton
The aquoeus solutions of 10%
NIPA/DMA monomer mixtuie with piedetennmed weight ratios of 90:10 and 85:15 were divided into clean glass ampoules of 5 mL, degassed by bubbling with nitrogen gas for 10 min, sealed off for sampling Each sample was irradiated at 20 kOy under gamma ray at Hanoi biadiation Center as described m our Plovious studies [8], Absoibed dose was measured with ECB dosuneters attached to the salqiles. After irradiation, the obtamed P(NIPAAm^X)-DMA) hydrogels were collected and cut mto 1 cm length, -The hydrogel disks were immeiged to excess distilled water at 60°C for a week to remove non.crossUnked polymers/monomera, dried at room temperahue overnight, then under vacuum,
C. Measurements
Swelling degree of hydrogel
Dried hydmgel disks were immeised ta distilled water in glass beaker, which were put in water badi at room temperature (25 ± 0,2°C), for 72 h to reach the equilibrium of sweUing After that, the swollen hydrogel was taken, blotted out the excess water usmg paper tissue,
41
and then weighed directly. Water sweUing ratios of hydrogels were calculated as follow:
Water swelling ratio
wheremoandnisarelheweightsofinitial and swollen hydrogel, hi this experiment, each vahie was calculated as an average of three separate measurements.
Drug loading and conlrol release tesl
The resulting P(NlPAAm<:o-DMA) hydrogeU were loaded widi dexamethasone or tegafur by solution loadtag method [14],hi which, a certam amount of dried hydrogel (W^|
was equihbraled m an aqueous solution contammg 5000 ppm of the drug at room temperature [15], After 72 h mcubaHon, the drug-loaded hydrogel (carrier) was taken out, washed with cold water. The optical absorijance of the solution with remaintag dmg was detennmed by a Shnnadzu UV-2450 spectrophotometer. The dmg concentration was extrapolated using the caUbration curve conespondmg to each drug. The loadmg dmg content and loadmg capacity were determined by diffeience ofthe mitial or alhninistered dose (Wa) and the lemaming (Wo.) dmg or residual dose to die drug solution (%) as foUowmg equations:
hydrogel)=
(%) = ^
loading drug content (mg/g
(2)capacity
(3)
About 50 mg drug cairieis were
transfened m a glass beaker contatatog 200 mL
of distdled water or simulated body fluid for the
m vttro release behavior smdies, AU beakers
were pm to shaker bath that keep at a ceriam
RADI.ATION PREPARATION OF DRLG CARRIERS FOR DEXAMHTHASONE AND TEGAFUR . temperature After predetermined time periods,
aliquots of 3 m l were uithdrawn and their optical absorbance \alues \\erc measured by UV spectrometer at 241 5 and 2^1 nm for dexamethasone and legafur. respectn ely The drug release was reported as percentage release as follow
% drug release al i nine — IOO
where Dm and D|,^,ui are the weight of released drug ai time t and total drug loaded in the drug cames,
//( vitro skin irritation test
Biocompatibility of the drug camers were analyzed by the in yiiro skin imtation test, followed OECD standard tesi guideline TG 404 ofthe National Instimte of drug quality control.
For this, the drug loaded hydrogels is applied in a single dose lo a small area of skin {- 6 cm^) of an experimental animal (rat), untreated skin area ofthe test animal serve as the control
kG\ was chosen tor radiation preparation ofthe hvdrogels But the initial ratio of monomers also affected to their morphology as observed in the Figure (Ic & d). This is due to the mobilitv' of network increased with the increasing of hydrophdie constituent of DMA domain in the hydrogels [15]
B. Swelling behavior
(4)
NIPAM/DMASO'lO
NIPAM/DMA 85/15
0 10 20 30 i Incubation lime (h)
:. Swelling behavior of P(NIPAAm-co-DMA) hydrogels
Fig. 1, Resulting hydrogels from lhe irradiated solutions al swollen (A); dried state (B). and SEM .mages of hydrogels from 90 10 (C) and 85 15 NIPA/DMA by weight ratio (D)
III. RESULTS AND DISCUSSIONS A. Radiation preparation o f t h e P(NIPAAni- co-DMA) hydrogels
Figure I showed an example of P(NlPAAm-co-DMA) hydrogels obtained from the solutions of 90' 10 and S5 15 of NIPA.'DMA (x\ u ) b> gamma irradiation As described m our previous stud>. lhe radiation dose of 20
Water swelling behaviors of P(NIPAAm- co-DMA) hydrogels were investigated at room temperature. As presented in Figure 2, swelling degrees of all hydrogels increased with mcubation lime, reach ti an equilibrium swelling values of aboul 13 and 18 g'g for the hydrogels obtained from NIPA/DMA of 90-10 and 85:15, respectively These swelling ratios are rather high suggested that Ihe hydiugels can
w HOANG OANG SANG et al.
also absoib lai;^,^ mima of Aug orftnher be moilifled to become eSfective conlioltoilwtase
•^amas. -The sweUing degiee of h y t t a ^ also
""^frased with DMA, a l^Jdn^hUic couslitoait m the hydrogel as mertioned 4i.our last s W y [11], This may he due 10 a Mglto'water content
Table rCplateaadlbaiiiug capacity of desamediasone by the hydrogel
swoUen by hydrophilic inteiaction of DMA with water,
C Fonnation of a dnig carrier and dmg loading
hydroXtL^rtoi^TLi:!" rrr-^r^r-""'"^-*
— to T ^ : and Table I. £ d ™ ^ t T . ^ ^ : ^ . X T . ' ^ ^ t . m prachce are lower thau that tegafiu, respectively.
Tablen. Uptake and loading capacity of tegafur by Uic hydrogel
Maxunum amounts of the loaded drugs were 48,6 and 95,7 mg/g dried hydrogels for dexamelhasone and tegafur, respectively, hi the same condidous, tegaflir showed hi^er loadtog capacity. The amount of uicoiporated dmg and fateractiou type of diug and hydrogels much depend ou die struchires and propeities of both drug and hydrogel. These results may he explained by die higher water sohibiUty of tegafur. However, furdier studies should be
carried out to esdmate dieir applicabihty to practice,
D. In vitro drug release
The drug release ftom the hydrogel was
measured widi mcubation Hme, and Figure 3
showed die cmnuladve release of drug as
flmcdons of tfaK at 25, 37 and W C It was
observed diat die release rates were qmte 6st
for bodi dmg models, and most dmgs released
R.\Dl.ATIO\ PREP-XR_-\TIO\ OF DRI G CARRIERS FOR DE.XAMETHASONE AND TEGAFUR .
0 0
BO
6 0
2 0
B
t
y
* 4 0 C 37 CFig. 3. Cumulatne release of dexamelhasone (A) and tegaflir (B) trom P(MA.'\m-co-DMA.) hydrogels in simulated body fluid at various temperamres from the hydrogels after first 24 h After that,
the drug seemed not release any more for legafur or released at \ ery slow rate for dexamethasone. At the same temperature, dexamethasone gradually released al first 12 h, slower released after 24 h, and then equilibrated after 36 n, whereas legafur quickly release during first 12 h, then reach to equilibnum state.
The release of drug through hydrogels may cause by diffusion of the drug trapped on hydrogels, morphological, structural and characteristic changes in hydrogel, degradation of polymer etc. However, it is very difficult to predict the drug release, because it was governed by some parameters such as nature of drug, degradabihty of polymer hydrogels, polymer-drug interaction
Figure 3 also revealed that the content of lelcased drugs reduced with increasing of the solution temperature. This is due to the hydrogels shrinking when heated through their LCST. when the drug difiused from inside to surface, but do not released from the hydrogels As the result, the drug release reduced by heatmg. There is a large amount of drug still remamed in the hydrogels after 36 h immersed m the simulated human body tluid This may due to a fact that both drug models are more or less h\drophilic, and the hydrophilic interaction rre\onI the drug release from the hydrogels
3.5. Biocompatibility
In Vitro skin irritation test showed no any skin irntation can be observed in the tested ammals during investigation period, suggesting that the dexamethasone and tegafur loaded P(NlPAAm-co-DMA) hydrogels can be applied as the drug camers fbr ftirther experiments
IV. CONCLUSION Two kmds of P(NIAPAm-co-DMA) hydrogels were prepared by simultaneously gamiTia radiadon copolymerization and crosslinking The resulting hydrogels were thermo-sensitive with swelling behavior that are suitable to use as drug camers and their equilibrium swelling degree increased with the increase of the hydrophilic segirients of DMA m the initial solution
The drug models were easily incorporated into the hydrogels followed solution loading method by the same loadmg process The drug-loaded hydrogels can be applied as drug carriers and their loadmg capacities were 48,6 and 94 7 mg/g (w/w) dried hydrogel for dexamethasone and tegafur.
respectively
In vitro release test showed that drug release from the camers of dried P(NlPA-co- DMA) hydrogels were quite fast, and the
HOANG DANG SANG ct aL release of tegafiic was a s t e r dian diat o f
* ™ ™ « h a s o n e m distiUed water as well as simulated human body fluid. The results also revealed diat die d m g release rate was
"i^Kndent on temperatnre,,'There are n o any skm irritation caused b y hydrogels observed for the product, meam ttiat die hydrogels can b e apphed as subcutaneous or hypodetmic drag carriers,
A C K N O W L E D G E M E N T S W e w o u U Uke to fliank Hanoi Irradiadon Center and Vietaam Atomic Energy histitate for dieh- financial supports with die project of CS/14/08.01.
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