- Email: [email protected]
Development and evaluation of thermo-sensitive hydrogel system with nanocomplexes for prolonged subcutaneous delivery of enoxaparin
Accepted Manuscript Development and evaluation of thermo-sensitive hydrogel system with nanocomplexes for prolonged subcutaneous delivery of enoxaparin Guihua Fang, Jing Zhou, Yu Qian, Jingxin Gou, Xiang Yang, Bo Tang PII:
S1773-2247(18)30735-4
DOI:
10.1016/j.jddst.2018.09.004
Reference:
JDDST 765
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 6 July 2018 Revised Date:
16 August 2018
Accepted Date: 2 September 2018
Please cite this article as: G. Fang, J. Zhou, Y. Qian, J. Gou, X. Yang, B. Tang, Development and evaluation of thermo-sensitive hydrogel system with nanocomplexes for prolonged subcutaneous delivery of enoxaparin, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/ j.jddst.2018.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development and evaluation of thermo-sensitive hydrogel system with
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nanocomplexes for prolonged subcutaneous delivery of enoxaparin
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Guihua Fang1, Jing Zhou1, Yu Qian1, Jingxin Gou2, Xiang Yang1, Bo Tang*1
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226001, China
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Shenyang, Liaoning Province, 10016, China
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School of Pharmacy, Nantong University, 19 Qixiu Road, Nantong, Jiangsu Province,
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Tel: +86 - 0513-85051728
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E-mail: [email protected]
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Corresponding author: Bo Tang (B. Tang)
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School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,
Abstract
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Here we report the combination of thermo-sensitive hydrogel systems and
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nanocomplexes
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Thermo-sensitive hydrogels were prepared with enoxaparin solution and dispersion of
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enoxaparin nanocomplexes. Nanocomplexes (NC) were formed by self-assembly of
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enoxaparin with cationic polymers. Three polymers namely ε-polylysine (Plys),
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chitosan (CS), polyethylenimine (PEI), were tested. Three nanocomplexes were
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optimized in terms of different EX/polymer mass ratio, and their size, zeta-potential
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and morphology were evaluated. Thermo-sensitive hydrogels were examined by
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gelation temperature, gel dissolution. In vitro EX release study demonstrated that
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nanocomplexes incorporation into thermo-sensitive hydrogels could prolong the EX
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release. Overall, nanocomplexes in thermo-sensitive hydrogels is promising delivery
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systems for prolonged subcutaneous EX delivery.
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Key words: thermo-sensitive hydrogel; nanocomplexes; subcutaneous delivery;
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enoxaparin; In vitro release
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subcutaneous
delivery of enoxaparin
(EX).
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1. Introduction Heparin, a highly sulfated natural polysaccharide, has been successfully used in
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the treatment of deep venous thromboembolism (DVT), venous thrombosis and
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pulmonary embolism (PE) [1]. However, it has been replaced by low molecular
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weight heparin (LMWH), such as enoxaparin, due to its severe clinical side effects
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and short elimination half life [2]. Enoxaparin is obtained by depolymerization of
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heparin with chemical methods ranging molecular weight from 3.8 kDa to 5 kDa [3].
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Generally, it is administered by subcutaneous route, and metabolized by liver and
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kidney with half life of about 4.5 h [4, 5]. Though the dosing frequency of enoxaparin
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is reduced than heparin, it still required once daily injection or twice daily injection
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[6]. Therefore, it’s necessary to develop new drug delivery systems to prolong the
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subcutaneous delivery of enoxaparin.
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Injectable depot formulations for long-term controlled drug release can help to
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reduce the frequency of administration, and lead to a number of successful
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pharmaceutical products [7]. As one of these injectable depots, in situ hydrogels have
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attracted an increasing interest for decades owing to its many advantages, including
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the simplicity of preparation, as well as convenient administration and improved
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patients compliance. Generally, in situ hydrogels are divided into two classes based
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on the gelation mechanism: chemically crosslinked in situ hydrogels and physically
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crosslinked in situ hydrogels [8]. Compared with chemically crosslinked in situ
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hydrogels, injectable physically crosslinked in situ hydrogels posses many advantages.
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On one hand, physically crosslinked in situ hydrogels can avoid using small molecule
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cross-linkers, which is not only detrimental to the tissue but also can destabilize the
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encapsulated drugs. On the other hand, the gelation time of physically crosslinked in
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situ hydrogels is much shorter than chemically crosslinked in situ hydrogels, which
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prevents the flow of polymers to other tissues and inhibits undesired drug leakage [9].
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Different physical stimulus including temperature [9-12], pH [13, 14], enzymes [15]
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and light [10] can result in in situ hydrogel formation. Temperature is the most
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commonly used stimulus in environmentally responsive systems. The change of
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temperature is not only relatively easy to control, but also easily applicable both in
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temperature and become a non-flowing gel at body temperature, are formed by a
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simple phase transition (sol-gel transition) in water without any chemical reaction and
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the process is reversible [16]. As such, thermo-sensitive hydrogels can serve as an
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injectable implant and thus a barrier for the release of the loaded drug.
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Pluronic® F127 (F127) or Poloxamer P407 is one of the most widely used for
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preparation of thermo-sensitive hydrogels for delivery of hydrophilic or hydrophobic
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drugs. It has been approved by the FDA and considered to be non-toxic. F127
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hydrogels is studied as topical drug delivery carrier by different administration routes
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such as subcutaneous [12, 17], intramuscular [18], ocular [19], nasal [20] and rectal
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[21]. Despite of many application advantages of F127 hydrogels, its subcutaneous
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longevity is too short, usually less than 3 days [22]. Therefore, the sustained effect of
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enoxaparin is not quite satisfactory.
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In order to solve the problem, two strategies have recently been applied to
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prolong the release of drug within subcutaneous injection site. One approach involves
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the synthesis of novel thermo-sensitive polymers [23]. Yet another approach involves
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development of particle/hydrogel combination system that entrapment nanoparticles,
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liposomes, or microspheres in a thermo-sensitive hydrogels [24]. For the synthesis of
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new polymers, it involves organic solvents, and residual solvents are harmful to the
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health. For the preparation of particle/hydrogel combination system, we can choose
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proper method to avoid organic solvents. Polyelectrolyte nanocomplexes are formed
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by self-assemble of positive- with negative-polyelectrolyte. Enoxaparin possesses
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high anionic charge, positive polymer can be used to mix with it to form
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polyelectrolyte nanocomplexes. Such method has the advantage of not necessitating
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organic solvents during preparation, therefore reducing possible damage to health.
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In this study, we considered to combine nanocomplexes and hydrogels for longer
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sustained drug delivery. Three polymers namely ε-polylysine (Plys), chitosan (CS),
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polyethylenimine (PEI) were used to prepare the water-soluble nanocomplexes of
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enoxaparin. The nanocomplexes were mixed with F127 to prepare F127 hydrogels
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incorporating the nanocomplexes of EX and Plys/CS/PEI. The in vitro EX release
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profile from the hydrogels was examined.
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2. Materials and Methods
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2.1 Materials Enoxaparin (mean MW 4251 Da) was purchased from Hangzhou Jiuyuan Gene
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Engineering Co., Ltd. (Hangzhou, China). Chitosan (300 kDa) was purchased from
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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) with a degree of
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deacetylation (DD) of 83.4%, ε-polylysine was purchased from Best-Reagent Co., Ltd.
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(Chengdu, Sichuan) with 25-30-lysine residues, Polyethylenimine (10 kDa) was
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purchased from Aladdin (Shanghai, China). Pluronic F127 (BASF, Ludiwigshafen)
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was purchased from Xi’an Yuelai Medical technology Co., Ltd. (Xi’an, China). All
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other chemicals were of analytical grade.
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2.2 Preparation of nanocomplexes
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2.2.1
Preparation of EX/Plys nanocomplexes
To prepare the nanocomplexes of EX with Plys, the Plys and EX were dissolved in
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deionized water separately. EX solution (1mg/ml) was added into 2 ml of Plys
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solution (2mg/ml) dropwise with different mass ratio (1:4; 2:4; 3:4; 4:4; 5:4) under
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magnetic stirring, and incubated for further 30 min at room temperature.
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Preparation of EX/CS nanocomplexes
To prepare the nanocomplexes of EX with CS, the CS was dissolved in 1% acetic
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acid and the EX was dissolved in deionized water. EX solution (1mg/ml) was added
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into 2 ml of CS solution (2mg/ml) dropwise with different mass ratio (1:4; 2:4; 3:4;
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4:4; 5:4) under magnetic stirring, and incubated for further 30 min at room
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temperature.
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Preparation of EX/PEI nanocomplexes
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To prepare the nanocomplexes of EX with PEI, the PEI and EX were dissolved in
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deionized water. EX solution (1mg/ml) was added into 2 ml of PEI solution (1mg/ml)
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dropwise with different mass ratio (0.5:2; 1:2; 2:2; 3:2; 4:2) under magnetic stirring,
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and incubated for further 30 min at room temperature.
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2.3 Physicochemical characterization of nanocomplexes
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The particle size of the prepared nanocomplexes was measured by 90 plus zeta
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(Brookhaven, USA) with a scattering angle of 90°, and the zeta potential
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measurements were carried out using the 90 plus zeta by electrophoretic laser doppler
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anemometry at room temperature. The morphology of nanocomplexes was observed by transmission electron
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microscope. Samples of nanocomplexes were diluted with deionized water, dropped
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onto a copper grid and then negatively stained with 2% phosphotungstic acid. The
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samples were air-dried and examined.
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The encapsulation efficiency (EE) of nanocomplexes was determined by
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ultrafiltration method. Briefly, the nanocomplexes were placed into an ultrafiltration
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device with MWCO 100 kDa and then centrifuged at 3000 rpm for 15 min. The
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concentration of filtration was determined with Azure A colorimetric method. The EX
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encapsulation efficiency was calculated according to the following equation.
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2.4 Preparation and optimization of thermo-sensitive F127 hydrogels Formulation containing optimized concentration of F127 was used for further
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investigation. Thermo-sensitive F127 hydrogels were prepared by cold method.
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Briefly, for blank F127 hydrogels, the calculated amount of F127 was added to
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deionized water and maintained at 4 ℃ until homogeneous solution formed. For
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drug-loaded F127 hydrogels, the deionized water was replaced by EX solution or EX
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nanocomplex suspension. Optimization of blank F127 hydrogels was done by varying
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the concentration of F127 and evaluating them for gelation temperature.
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2.5 Gelation temperature of thermo-sensitive F127 hydrogels
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A vial inversion method was employed to determine the gelation temperature of
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thermo-sensitive F127 hydrogels [25]. In brief, 10 ml of gels was transferred to a vial,
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immersed in a water bath was increased at 0.5 ℃ for at least 5 minutes from 15 ℃ to
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50 ℃. The gelation temperature (Tsol-gel) was recorded after the gels would no longer
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move upon inversing the vials through an angle of 90°.
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2.6 Scanning electron microscope (SEM) of thermo-sensitive hydrogels The morphological feature of the four thermo-sensitive hydrogels (EX solution
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hydrogels, EX/Plys nanocomplexes hydrogels, EX/CS nanocomplexes hydrogels,
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EX/PEI nanocomplexes hydrogels) were characterized by an SEM (Hitach-S4800,
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Japan). The samples were frozen at -80 ℃ and lyophilized at -50 ℃ for 48 h. The dry
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samples were sputtered with gold before observation.
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2.7 Hydrogel dissolution
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When the F127 hydrogels administered by subcutaneous, they would be
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contacted with body fluids, such as extracellular fluid, resulting in gel dissolution. In
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order to simulate the hydrogel dissolution in vivo, phosphate buffer saline (pH 7.4)
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was used as a release medium. Hydrogel dissolution profiles of F127 hydrogels
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containing EX solution or EX nanocomplexes were examined using a membraneless
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model. Briefly, 3 ml each cold formulation was transferred into graduated glass tubes
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with stopper, and placed in a 37 ℃ water bath until a non-flowing gel was formed.
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Then, 2 ml of release medium preheated at 37 ℃ and covered the surface of gels. At
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predetermined time, tubes were gently turned up and down several times, and the
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remaining volume of gels in tubes was recorded. Meanwhile, 2 ml of release medium
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was withdrawn from a sample and replaced by an equal volume of the fresh release
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medium. Hydrogel dissolution volume was defined as the differences in volume of
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hydrogels between two time points.
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2.8 In vitro EX release study
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In vitro EX release study was conducted as previous described “Hydrogel
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dissolution” experiment. At the given time, 2 ml of release medium was taken out and
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supplemented with fresh release medium. The amount of EX released from hydrogels
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was determined according to Azure A colorimetric method. All release experiments
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were performed as triplicates.
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3.1
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Results and discussion Preparation and characterization of nanocomplexes In this study, self-assembled nanocomplexes were prepared by electrostatic
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polyethyleneimine) and the negatively charged enoxaparin. The structures of EX, Plys,
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CS and PEI are shown in Fig.1. This preparation method was green and solvent-free,
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so it’s quite safe for human use. Table 1 shows size, polydispersity index (PDI) and
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zeta potential of the nanocomplexes prepared at different mass ratio of EX and three
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polymers. The particle size of nanocomplexes decreased when the mass ratio of EX
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and Plys was increased from 1:4 to 3:4, and then increased when the ratio was
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increased from 3:4 to 5:4. In contrast, for EX/CS nanocomplexes, with an increase in
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proportion of EX, the particle size was decreased. A similar particle size change was
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observed for EX/PEI nanocomplexes. Additionally, the zeta potential of three
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nanocomplexes was decreased with an increase of proportion of EX, which is
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attributed to the excess presence of negatively charged carboxylic groups and sulfate
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groups on EX molecules. Furthermore, nanocomplexes with higher EX/polymer mass
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ratio (EX/Plys ≥ 4:4; EX/CS ≥ 4:4; EX/PEI ≥ 4:2) became unstable and tended to
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precipitate, which was probably due to the excess EX could not bind polymers tightly
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at a certain amount of polymers. Meanwhile, in order to prepare stable, homogenous
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and concentrated nanocomplexes, EX/Plys = 3:4, EX/CS = 3:4, and EX/PEI = 3:2
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were chosen as optimal mass ratio. Consequently, these nanocomplexes were used to
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fabricate the thermoreversible hydrogels.
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The three optimal nanocomplexes were visualized by TEM. The TEM images of
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nanocomplexes are shown in Fig.2, indicating that all nanocomplexes were spherical,
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and the sizes were similar to the results obtained by dynamic light scattering
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technique. In addition, the encapsulation efficiency of three optimal nanocomplexes
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was determined according to ultrafiltration method, and the EE for EX/Plys
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nanocomplexes, EX/CS nanocomplexes and EX/PEI nanocomplexes was 97.2%,
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98.5% and 98.2%, respectively, indicating that EX could almost completely bind
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these positively charged polymers.
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3.2 Optimization of concentration of F127
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Gelation temperature is the temperature at which the liquid phase undergoes the
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transition from solution to gel. Gelation temperature was determined according to the
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measured for the concentration range of 15%-24% (F1-F4). As shown in Table 2, it
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was found that gelation temperature of F127 hydrogels decreased with an increase of
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concentration of F127. In order to ensure that F127 hydrogels combined the
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advantages of convenient administration and long-acting drug depot, the prepared
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F127 hydogels should be liquid state at room temperature (25 ℃) and form semi-solid
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gels at body temperature (37 ℃). Hence, 21% (w/v) concentration of F127 was
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selected as for further studies. When drug solutions or nanocomplexes were added
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into F127 hydrogels (F5-F8), it was found that gelation temperature of formulations
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did not change, which suggested that incorporation of drug could not cause
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modification of the process of micellar association of F127 hydrogels.
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F127 copolymer blocks based on poly (ethylene oxide) - b - poly (propylene
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oxide) - poly (ethylene oxide) (PEO-PPO-PEO) sequences. Fig.3 illustrates the
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establishment of nanocomplexes in hydrogels and mechanism of gelation of F127.
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Namely, F127 forms micelles above critical micelles concentration (about 1 mg/ml)
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[26]. Further, below a lower critical solution temperature (< LCST), both ethylene and
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propylene oxide blocks are hydrated, and PPO is relatively soluble in water. As the
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temperature increases (> LCST), the polymer solution turns into a gel owing to the
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micelles packing and entanglements.
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3.3 Scanning electron microscope (SEM) of thermo-sensitive hydrogels
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SEM micrographs of lyophilized hydrogels structure were presented in Fig.4.
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From the micrographs, we can see that the structures of four thermo-sensitive
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hydrogels were almost the same, and the inner structures were porous with mesh size
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of about 1 µm. This probably could be attributed to the same F127 concentration for
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those hydrogels.
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3.4 Dissolution of hydrogels
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In general, F127 hydrogels undergo dissolution in an aqueous environment
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owing to the water penetration into gel network, leading to unpacking of the F127
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micelles, polymer hydration and finally hydrogels dissolution. The dissolution
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profiles of different F127 hydrogels are shown in Fig.5. From the above results, it can
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be concluded that hydrogels with EX solutions and EX nanocomplexes (EX/Plys
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nanocomplexes, EX/CS nanocomplexes, EX/PEI nanocomplexes) had similar
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dissolution profiles for the same concentration of F127.
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3.5 In vitro release of EX Because these hydrogels were intended for subcutaneous administration,
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membraneless method was utilized to evaluate the in vitro release of EX from F127 in
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situ hygrogels, which is to be closer to the in vivo condition. Fig. 6 indicates that
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27.78%, 13.01%, 13.98% and 9.02% of EX were cumulatively released from solution
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hydrogels, EX/Plys nanocomplex hydrogels, EX/CS nanocomplex hydrogels and
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EX/PEI nanocomplex hydrogels at 20 h, respectively. In contrast with EX solution
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hydrogels, there was a prolonged release of EX from the nanocomplexes hydrogels.
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Approximately 99.37% of the cumulative amount of EX was released from EX
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solution hydrogels within 144 h. In the case of EX/Plys nanocomplexes hydrogels,
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EX/CS nanocomplex hydrogels and EX/PEI nanocomplex hydrogels, the percent
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cumulative release of EX was only about 30.39%, 39.52% and 22.18% within 144 h.
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From above mentioned results, it can be concluded that EX nanocomplexes could
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prolong the release of EX from hydrogels.
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Three different release models were used to predict the drug release, and the
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regression results for the release of EX from hydrogels are shown in Table 3. The in
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vitro release of EX from solution hydrogels, EX/Plys nanocomplex hydrogels, EX/CS
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nanocomplex hydrogels and EX/PEI nanocomplex hydrogels corresponds to the
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Ritger-Peppas model with r values of 0.997, 0.983, 0.994, 0.991. Ritger-Peppas
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equation has three different meanings. One is in the case of n (release exponent) <
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0.45, indicating that drug release is diffusion-controlled, namely Fickian diffusion.
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Yet another is in case of n > 0.89, indicating that drug release is erosion-controlled.
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Values of n between 0.45 and 0.89 can be considered as an indicator for diffusion-
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and erosion- controlled dual release mechanism. From the Table 3, it can be
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concluded that drug released from solution hydrogels or nanocomplex hydrogels was
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controlled by diffusion and erosion. As we know, erosion is determined by F127
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hydrogels dissolution, and F127 hydrogels dissolution mainly depends on F127
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depends on the mesh sizes within the hydrogels matrix, but also on hydrodynamic
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radius of the drug molecules [27]. In our study, the mesh sizes almost were the same
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(Fig.4) due to the same concentration of F127 and preparation method. The size of EX
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solution and three nanocomplexes was smaller than 1 µm (hydrogels pore size)
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Therefore, for EX solution hydrogels, EX could diffuse from hydrogels freely along a
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concentration gradient. In addition, some EX would release while hydrogels
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dissolution. For EX nanocomplexes hydrogels, the release process was relatively
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complicated. Maybe three kind of processes co-exist. First, EX nanocomplexes also
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could diffuse from pores of hydrogels, and then EX released from nanocomplexes
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through nanocomplexes dissociation. Second, EX dissociated from nanocomplexes
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and then released from the hydrogels. Third, EX directly released from hydrogels with
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hydrogels dissolution. The first two may be the main reason why nanocomplexes
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prolong the release of EX in hydrogels. Besides, among three nanocomplexes, EX/CS
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nanocomplexes release rate was relatively faster than other two nanocomplexes.
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During the course of experiment, the pH of the EX/CS nanocomplexes system was
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gradually increasing after the addition of the fresh release medium (pH 7.4). Chitosan
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molecules deprotonated and precipitated, resulting in faster dissociation of
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nanocomplex and EX release due to chitosan deionization. For EX/Plys
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nanocomplexes and EX/PEI nanocomplexes, they keep stable in release medium, so
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EX released from the two groups was relatively slower. Also, EX released from
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EX/PEI nanocomplexes was the slowest, this probably was due to the strong
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electrostatic interaction between EX and PEI polymer, which has rather high charge
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density. From above all, it can be concluded that the combination of thermo-sensitive
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hydrogels and nanocomplexes could significantly sustain the drug release, and the
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drug release from nanocomplexes/hydrogels was controlled by hydrogels dissolution
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and nanocomplexes dissociation.
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3.6 Correlation between hydrogels dissolution and drug release
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To further investigate whether the difference observed in EX release between
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solution/hydrogels and nanocomplexes/hydrogels is due to the hydrogels dissolution
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and nanocomplexes dissociation, the correlation between the percentage of EX
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released and the percentage of hydrogels dissolved was linear fitted. As shown in
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Fig.7, it can be seen that good linear correlation between them, which indicated that
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theses
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Additionally, the linear order was solution (R² =0.9952) > EX/CS nanocomplex (R²
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=0.9867) > EX/Plys nanocomplex (R² =0.9720) > EX/PEI nanocomplex (R² =0.9636),
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which
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dissociation-controlled. Therefore, EX released from nanocomplex/hydrogels is both
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hydrogels dissolution- and nanocomplexes dissociation-controlled.
primarily
demonstrated
that
hydrogels
EX
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released
release.
from
nanocomplex
also
4. Conclusion
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dissolution-controlled
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In this study, we developed an EX nanocomplexes/F127 thermo-sensitive
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hydrogel composite system which possessed the same thermo-sensitive property as
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blank F127 hydrogels, and achieved prolonged drug release. Three positively-charged
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polymers were used to prepare EX polyelectrolyte nanocomplexes. In vitro release
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results indicated that nanocomplexes fabricated by different polymers held different
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release rates, and PEI polymer could preferably sustain the EX release. In order to
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clarify how the PEI affected the drug release from the hydrogel systems in detail,
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different concentration and molecular weight of PEI will be investigated in our study
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in future.
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Acknowledgements
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This work was supported by the Natural Science Fund for Colleges and Universities
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in Jiangsu Province (No. 17KJB350009) and Natural Science Foundation of Jiangsu
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Province (No. BK20170445).
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[12] M. Radivojsa Matanovic, I. Grabnar, M. Gosenca, P.A. Grabnar, Prolonged subcutaneous delivery of low molecular weight heparin based on thermoresponsive hydrogels with chitosan nanocomplexes: Design, in vitro evaluation, and cytotoxicity studies, International journal of pharmaceutics, 488 (2015)
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[14] J. Qu, X. Zhao, P.X. Ma, B. Guo, pH-responsive self-healing injectable hydrogel based on N-carboxyethyl chitosan for hepatocellular carcinoma therapy, Acta Biomaterialia, 58 (2017) 168-180. [15] D. Wlodarczyk, J.P. Méricq, L. Soussan, D. Bouyer, C. Faur, Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology, International Journal of Biological Macromolecules, 107 (2018) 1175-1183. [16] Y. Chen, Y. Li, W. Shen, K. Li, L. Yu, Q. Chen, J. Ding, Controlled release of liraglutide using thermogelling polymers in treatment of diabetes, Sci Rep, 6 (2016) 31593. [17] Y. Liu, W.-L. Lu, J.-C. Wang, X. Zhang, H. Zhang, X.-Q. Wang, T.-Y. Zhou, Q. Zhang, Controlled delivery of recombinant hirudin based on thermo-sensitive Pluronic® F127 hydrogel for subcutaneous administration: In vitro and in vivo characterization, Journal of Controlled Release, 117 (2007) 387-395. [18] K. Zhang, X. Shi, X. Lin, C. Yao, L. Shen, Y. Feng, Poloxamer-based in situ hydrogels for
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controlled delivery of hydrophilic macromolecules after intramuscular injection in rats, Drug Deliv, 22 (2015) 375-382. [19] K. Al Khateb, E.K. Ozhmukhametova, M.N. Mussin, S.K. Seilkhanov, T.K. Rakhypbekov, W.M. Lau, V.V. Khutoryanskiy, In situ gelling systems based on Pluronic F127/Pluronic F68 formulations for ocular drug delivery, International journal of pharmaceutics, 502 (2016) 70-79. [20] M.J. Bhandwalkar, A.M. Avachat, Thermoreversible nasal in situ gel of venlafaxine hydrochloride: formulation, characterization, and pharmacodynamic evaluation, AAPS PharmSciTech, 14 (2013)
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[22] Z. Lin, W. Gao, H. Hu, K. Ma, B. He, W. Dai, X. Wang, J. Wang, X. Zhang, Q. Zhang, Novel thermo-sensitive hydrogel system with paclitaxel nanocrystals: High drug-loading, sustained drug release and extended local retention guaranteeing better efficacy and lower toxicity, Journal of Controlled Release, 174 (2014) 161-170.
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[23] P. Wang, W. Chu, X. Zhuo, Y. Zhang, J. Gou, T. Ren, H. He, T. Yin, X. Tang, Modified PLGA–PEG–PLGA thermosensitive hydrogels with suitable thermosensitivity and properties for use in a drug delivery system, Journal Of Materials Chemistry B, (2017) 1551-1565. [24] M. Pitorre, H. Gondé, C. Haury, M. Messous, J. Poilane, D. Boudaud, E. Kanber, G.A. Rossemond Ndombina, J.-P. Benoit, G. Bastiat, Recent advances in nanocarrier-loaded gels: Which drug delivery technologies against which diseases?, Journal of Controlled Release, 266 (2017) 140-155.
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[25] P. Wang, Q. Wang, T. Ren, H. Gong, J. Gou, Y. Zhang, C. Cai, X. Tang, Effects of Pluronic F127-PEG multi-gel-core on the release profile and pharmacodynamics of Exenatide loaded in PLGA microspheres, Colloids and Surfaces B: Biointerfaces, 147 (2016) 360-367. [26] M.R. Matanović, J. Kristl, P.A. Grabnar, Thermoresponsive polymers: Insights into decisive hydrogel characteristics, mechanisms of gelation, and promising biomedical applications, International
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ACCEPTED MANUSCRIPT Table 1 Physicochemical characteristics of nanocomplexes at various EX/polymer mass ratios.
2:4 3:4 4:4 5:4
EX/CS
1:4 2:4 3:4
1:2
3:2
4:2
194.93±3.11
0.109±0.025
32.38±1.67
143.70±4.13
0.143±0.033
184.53±2.64
0.110±0.008
804.36±72.35
0.141±0.104
588.72±19.37
0.218±0.022
59.12±1.95
519.31±1.36
0.241±0.032
57.00±1.28
0.240±0.017
52.76±1.40
0.282±0.035
48.84±1.16
302.27±4.85
0.207±0.038
47.31±2.15
1082.54±40.01
0.377±0.043
7.70±0.77
442.20±79.73
0.325±0.039
4.69±2.75
319.91±6.10
0.172±0.049
-14.59±1.73
146.32±2.23
0.173±0.029
-22.94±0.79
114.11±0.687
0.272±0.007
-38.82±5.13
325.14±12.49
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2:2
39.96±0.30
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0.5:2
0.357±0.056
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EX/PEI
1091.06±469.36
386.61±0.60
4:4 5:4
Zeta potential (mV)
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1:4
Polydispersity index
27.96±2.53
23.43±2.21
-26.91±2.21
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EX/Plys
Size (nm)
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Table 2 Results of optimization of concentration of F127. Formulation batch
F1
F2
EX (mg/ml) Polymer (mg/ml) F127(%,w/v) Tsol-gel ( )
0 0 15
0 0 18 37
﹥60
F3
F4
F5*
F6*
F7*
F8*
0 0 21 28
0 0 24 22
0.6 0 21 28
0.6 0.4 21 28
0.6 0.4 21 28
0.6 0.4 21 28
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Tsol-gel, gelation temperature;
*, F5-F8 respectively represents EX solution hydrogels, EX/Plys, EX/CS, EX/PEI nanocomplexes
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Table 3
Release kinetics of EX solutions, EX/Plys nanocomplexes, EX/CS nanocomplex and EX/PEI nanocomplex hydrogels.
Zero order
First order
Ritger-Peppasa
Q versus t
ln (1 − Q) versus t
lnQ versus lnt
EX solutions hydrogels
y = 0.664x + 9.591
y = -0.664x + 90.409
y = 0.709x + 1.121
EX/Plys nanocomplexes hydrogels
r = 0.985 y = 0.180x + 5.888
r = 0.985 y = -0.180x + 94.113
r = 0.997 y = 0.517x + 0.859
EX/CS nanocomplex hydrogels
r = 0.947 y = 0.236x +6.120
r = 0.947 y = -0.236x + 93.880
r = 0.983 y = 0.540x + 0.947
r = 0.966 y = 0.137x +4.267 r = 0.952
r = 0.966 y = -0.137x + 95.733 r = 0.952
r = 0.994 y = 0.5201x + 0.553 r = 0.991
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Formulation
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EX/PEI nanocomplex hydrogels
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Ritger-Peppas equation: lnQ = nlnt + lnk, Q is the fractional drug release, n is the release exponent, t is the release time and k is a rate constant.
ACCEPTED MANUSCRIPT Figure captions Fig.1. Structure of (a) enoxaparin, (b) polylysine, (c) chitosan and (d) polyethyleneimine. Fig.2. TEM micrograph of (a) EX/Plys nanocomplexes, (b) EX/CS nanocomplexes
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and (c) EX/PEI nanocomplexes. Fig.3. Schematic structure of nanocomplex in F127 hydrogels. LCST: low critical solution temperature.
Fig.4. SEM micrograph of (a) EX solution in hydrogels, (b) EX/Plys nanocomplexes
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in hydrogels, (c) EX/CS nanocomplexes in hydrogels and (d) EX/PEI nanocomplexes in hydrogels.
(Ph 7.4, 37
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Fig.5. Dissolution profiles of thermoreversible hydrogels at physiological conditions ). Data are means ±SD of three measurements.
Fig.6. Release profiles of EX from F127 hydrogels (21%). Data are means ±SD of three measurements.
Fig.7. A correlation of cumulative percent of EX released with cumulative percent of
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F127 hydrogels. The line represents a linear regression. (a) EX solution in hydrogels, (b) EX/Plys nanocomplexes in hydrogels, (c) EX/CS nanocomplexes in hydrogels and
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(d) EX/PEI nanocomplexes in hydrogels.
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polyethyleneimine.
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Fig.1. Structure of (a) enoxaparin, (b) polylysine, (c) chitosan and (d)
Fig.2. TEM micrograph of (a) EX/Plys nanocomplexes, (b) EX/CS nanocomplexes and (c) EX/PEI nanocomplexes.
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Fig.3. Schematic structure of nanocomplexes in F127 hydrogels. LCST: low critical
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solution temperature.
Fig.4. SEM micrograph of (a) EX solution in hydrogels, (b) EX/Plys nanocomplexes in hydrogels, (c) EX/CS nanocomplexes in hydrogels and (d) EX/PEI nanocomplexes in hydrogels.
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Fig.5. Dissolution profiles of thermoreversible hydrogels at physiological conditions ). Data are means ±SD of three measurements.
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(Ph 7.4, 37
Fig.6. Release profiles of EX from F127 hydrogels (21%). Data are means ±SD of three measurements.
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Fig.7. A correlation of cumulative percent of EX released with cumulative percent of F127 hydrogels. The line represents a linear regression. (a) EX solution in hydrogels,
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(b) EX/Plys nanocomplexes in hydrogels, (c) EX/CS nanocomplexes in hydrogels and
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(d) EX/PEI nanocomplexes in hydrogels.
S1773-2247(18)30735-4
DOI:
10.1016/j.jddst.2018.09.004
Reference:
JDDST 765
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 6 July 2018 Revised Date:
16 August 2018
Accepted Date: 2 September 2018
Please cite this article as: G. Fang, J. Zhou, Y. Qian, J. Gou, X. Yang, B. Tang, Development and evaluation of thermo-sensitive hydrogel system with nanocomplexes for prolonged subcutaneous delivery of enoxaparin, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/ j.jddst.2018.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development and evaluation of thermo-sensitive hydrogel system with
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nanocomplexes for prolonged subcutaneous delivery of enoxaparin
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Guihua Fang1, Jing Zhou1, Yu Qian1, Jingxin Gou2, Xiang Yang1, Bo Tang*1
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1
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226001, China
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2
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Shenyang, Liaoning Province, 10016, China
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*
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School of Pharmacy, Nantong University, 19 Qixiu Road, Nantong, Jiangsu Province,
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Tel: +86 - 0513-85051728
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E-mail: [email protected]
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Corresponding author: Bo Tang (B. Tang)
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School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,
Abstract
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Here we report the combination of thermo-sensitive hydrogel systems and
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nanocomplexes
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Thermo-sensitive hydrogels were prepared with enoxaparin solution and dispersion of
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enoxaparin nanocomplexes. Nanocomplexes (NC) were formed by self-assembly of
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enoxaparin with cationic polymers. Three polymers namely ε-polylysine (Plys),
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chitosan (CS), polyethylenimine (PEI), were tested. Three nanocomplexes were
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optimized in terms of different EX/polymer mass ratio, and their size, zeta-potential
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and morphology were evaluated. Thermo-sensitive hydrogels were examined by
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gelation temperature, gel dissolution. In vitro EX release study demonstrated that
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nanocomplexes incorporation into thermo-sensitive hydrogels could prolong the EX
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release. Overall, nanocomplexes in thermo-sensitive hydrogels is promising delivery
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systems for prolonged subcutaneous EX delivery.
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Key words: thermo-sensitive hydrogel; nanocomplexes; subcutaneous delivery;
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enoxaparin; In vitro release
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subcutaneous
delivery of enoxaparin
(EX).
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for prolonged
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1. Introduction Heparin, a highly sulfated natural polysaccharide, has been successfully used in
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the treatment of deep venous thromboembolism (DVT), venous thrombosis and
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pulmonary embolism (PE) [1]. However, it has been replaced by low molecular
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weight heparin (LMWH), such as enoxaparin, due to its severe clinical side effects
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and short elimination half life [2]. Enoxaparin is obtained by depolymerization of
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heparin with chemical methods ranging molecular weight from 3.8 kDa to 5 kDa [3].
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Generally, it is administered by subcutaneous route, and metabolized by liver and
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kidney with half life of about 4.5 h [4, 5]. Though the dosing frequency of enoxaparin
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is reduced than heparin, it still required once daily injection or twice daily injection
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[6]. Therefore, it’s necessary to develop new drug delivery systems to prolong the
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subcutaneous delivery of enoxaparin.
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Injectable depot formulations for long-term controlled drug release can help to
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reduce the frequency of administration, and lead to a number of successful
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pharmaceutical products [7]. As one of these injectable depots, in situ hydrogels have
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attracted an increasing interest for decades owing to its many advantages, including
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the simplicity of preparation, as well as convenient administration and improved
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patients compliance. Generally, in situ hydrogels are divided into two classes based
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on the gelation mechanism: chemically crosslinked in situ hydrogels and physically
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crosslinked in situ hydrogels [8]. Compared with chemically crosslinked in situ
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hydrogels, injectable physically crosslinked in situ hydrogels posses many advantages.
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On one hand, physically crosslinked in situ hydrogels can avoid using small molecule
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cross-linkers, which is not only detrimental to the tissue but also can destabilize the
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encapsulated drugs. On the other hand, the gelation time of physically crosslinked in
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situ hydrogels is much shorter than chemically crosslinked in situ hydrogels, which
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prevents the flow of polymers to other tissues and inhibits undesired drug leakage [9].
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Different physical stimulus including temperature [9-12], pH [13, 14], enzymes [15]
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and light [10] can result in in situ hydrogel formation. Temperature is the most
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commonly used stimulus in environmentally responsive systems. The change of
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temperature is not only relatively easy to control, but also easily applicable both in
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ACCEPTED MANUSCRIPT vitro and in vivo. Thermo-sensitive hydrogels, which exist as flowing fluid at low
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temperature and become a non-flowing gel at body temperature, are formed by a
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simple phase transition (sol-gel transition) in water without any chemical reaction and
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the process is reversible [16]. As such, thermo-sensitive hydrogels can serve as an
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injectable implant and thus a barrier for the release of the loaded drug.
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Pluronic® F127 (F127) or Poloxamer P407 is one of the most widely used for
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preparation of thermo-sensitive hydrogels for delivery of hydrophilic or hydrophobic
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drugs. It has been approved by the FDA and considered to be non-toxic. F127
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hydrogels is studied as topical drug delivery carrier by different administration routes
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such as subcutaneous [12, 17], intramuscular [18], ocular [19], nasal [20] and rectal
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[21]. Despite of many application advantages of F127 hydrogels, its subcutaneous
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longevity is too short, usually less than 3 days [22]. Therefore, the sustained effect of
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enoxaparin is not quite satisfactory.
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In order to solve the problem, two strategies have recently been applied to
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prolong the release of drug within subcutaneous injection site. One approach involves
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the synthesis of novel thermo-sensitive polymers [23]. Yet another approach involves
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development of particle/hydrogel combination system that entrapment nanoparticles,
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liposomes, or microspheres in a thermo-sensitive hydrogels [24]. For the synthesis of
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new polymers, it involves organic solvents, and residual solvents are harmful to the
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health. For the preparation of particle/hydrogel combination system, we can choose
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proper method to avoid organic solvents. Polyelectrolyte nanocomplexes are formed
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by self-assemble of positive- with negative-polyelectrolyte. Enoxaparin possesses
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high anionic charge, positive polymer can be used to mix with it to form
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polyelectrolyte nanocomplexes. Such method has the advantage of not necessitating
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organic solvents during preparation, therefore reducing possible damage to health.
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In this study, we considered to combine nanocomplexes and hydrogels for longer
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sustained drug delivery. Three polymers namely ε-polylysine (Plys), chitosan (CS),
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polyethylenimine (PEI) were used to prepare the water-soluble nanocomplexes of
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enoxaparin. The nanocomplexes were mixed with F127 to prepare F127 hydrogels
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incorporating the nanocomplexes of EX and Plys/CS/PEI. The in vitro EX release
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profile from the hydrogels was examined.
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2. Materials and Methods
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2.1 Materials Enoxaparin (mean MW 4251 Da) was purchased from Hangzhou Jiuyuan Gene
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Engineering Co., Ltd. (Hangzhou, China). Chitosan (300 kDa) was purchased from
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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) with a degree of
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deacetylation (DD) of 83.4%, ε-polylysine was purchased from Best-Reagent Co., Ltd.
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(Chengdu, Sichuan) with 25-30-lysine residues, Polyethylenimine (10 kDa) was
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purchased from Aladdin (Shanghai, China). Pluronic F127 (BASF, Ludiwigshafen)
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was purchased from Xi’an Yuelai Medical technology Co., Ltd. (Xi’an, China). All
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other chemicals were of analytical grade.
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2.2 Preparation of nanocomplexes
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2.2.1
Preparation of EX/Plys nanocomplexes
To prepare the nanocomplexes of EX with Plys, the Plys and EX were dissolved in
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deionized water separately. EX solution (1mg/ml) was added into 2 ml of Plys
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solution (2mg/ml) dropwise with different mass ratio (1:4; 2:4; 3:4; 4:4; 5:4) under
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magnetic stirring, and incubated for further 30 min at room temperature.
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2.2.2
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Preparation of EX/CS nanocomplexes
To prepare the nanocomplexes of EX with CS, the CS was dissolved in 1% acetic
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acid and the EX was dissolved in deionized water. EX solution (1mg/ml) was added
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into 2 ml of CS solution (2mg/ml) dropwise with different mass ratio (1:4; 2:4; 3:4;
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4:4; 5:4) under magnetic stirring, and incubated for further 30 min at room
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temperature.
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2.2.3
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Preparation of EX/PEI nanocomplexes
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To prepare the nanocomplexes of EX with PEI, the PEI and EX were dissolved in
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deionized water. EX solution (1mg/ml) was added into 2 ml of PEI solution (1mg/ml)
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dropwise with different mass ratio (0.5:2; 1:2; 2:2; 3:2; 4:2) under magnetic stirring,
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and incubated for further 30 min at room temperature.
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2.3 Physicochemical characterization of nanocomplexes
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The particle size of the prepared nanocomplexes was measured by 90 plus zeta
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(Brookhaven, USA) with a scattering angle of 90°, and the zeta potential
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measurements were carried out using the 90 plus zeta by electrophoretic laser doppler
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anemometry at room temperature. The morphology of nanocomplexes was observed by transmission electron
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microscope. Samples of nanocomplexes were diluted with deionized water, dropped
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onto a copper grid and then negatively stained with 2% phosphotungstic acid. The
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samples were air-dried and examined.
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The encapsulation efficiency (EE) of nanocomplexes was determined by
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ultrafiltration method. Briefly, the nanocomplexes were placed into an ultrafiltration
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device with MWCO 100 kDa and then centrifuged at 3000 rpm for 15 min. The
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concentration of filtration was determined with Azure A colorimetric method. The EX
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encapsulation efficiency was calculated according to the following equation.
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2.4 Preparation and optimization of thermo-sensitive F127 hydrogels Formulation containing optimized concentration of F127 was used for further
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investigation. Thermo-sensitive F127 hydrogels were prepared by cold method.
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Briefly, for blank F127 hydrogels, the calculated amount of F127 was added to
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deionized water and maintained at 4 ℃ until homogeneous solution formed. For
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drug-loaded F127 hydrogels, the deionized water was replaced by EX solution or EX
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nanocomplex suspension. Optimization of blank F127 hydrogels was done by varying
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the concentration of F127 and evaluating them for gelation temperature.
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2.5 Gelation temperature of thermo-sensitive F127 hydrogels
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A vial inversion method was employed to determine the gelation temperature of
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thermo-sensitive F127 hydrogels [25]. In brief, 10 ml of gels was transferred to a vial,
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immersed in a water bath was increased at 0.5 ℃ for at least 5 minutes from 15 ℃ to
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50 ℃. The gelation temperature (Tsol-gel) was recorded after the gels would no longer
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move upon inversing the vials through an angle of 90°.
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2.6 Scanning electron microscope (SEM) of thermo-sensitive hydrogels The morphological feature of the four thermo-sensitive hydrogels (EX solution
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hydrogels, EX/Plys nanocomplexes hydrogels, EX/CS nanocomplexes hydrogels,
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EX/PEI nanocomplexes hydrogels) were characterized by an SEM (Hitach-S4800,
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Japan). The samples were frozen at -80 ℃ and lyophilized at -50 ℃ for 48 h. The dry
152
samples were sputtered with gold before observation.
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2.7 Hydrogel dissolution
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When the F127 hydrogels administered by subcutaneous, they would be
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contacted with body fluids, such as extracellular fluid, resulting in gel dissolution. In
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order to simulate the hydrogel dissolution in vivo, phosphate buffer saline (pH 7.4)
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was used as a release medium. Hydrogel dissolution profiles of F127 hydrogels
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containing EX solution or EX nanocomplexes were examined using a membraneless
159
model. Briefly, 3 ml each cold formulation was transferred into graduated glass tubes
160
with stopper, and placed in a 37 ℃ water bath until a non-flowing gel was formed.
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Then, 2 ml of release medium preheated at 37 ℃ and covered the surface of gels. At
162
predetermined time, tubes were gently turned up and down several times, and the
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remaining volume of gels in tubes was recorded. Meanwhile, 2 ml of release medium
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was withdrawn from a sample and replaced by an equal volume of the fresh release
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medium. Hydrogel dissolution volume was defined as the differences in volume of
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hydrogels between two time points.
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2.8 In vitro EX release study
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In vitro EX release study was conducted as previous described “Hydrogel
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dissolution” experiment. At the given time, 2 ml of release medium was taken out and
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supplemented with fresh release medium. The amount of EX released from hydrogels
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was determined according to Azure A colorimetric method. All release experiments
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were performed as triplicates.
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3
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3.1
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Results and discussion Preparation and characterization of nanocomplexes In this study, self-assembled nanocomplexes were prepared by electrostatic
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polyethyleneimine) and the negatively charged enoxaparin. The structures of EX, Plys,
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CS and PEI are shown in Fig.1. This preparation method was green and solvent-free,
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so it’s quite safe for human use. Table 1 shows size, polydispersity index (PDI) and
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zeta potential of the nanocomplexes prepared at different mass ratio of EX and three
182
polymers. The particle size of nanocomplexes decreased when the mass ratio of EX
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and Plys was increased from 1:4 to 3:4, and then increased when the ratio was
184
increased from 3:4 to 5:4. In contrast, for EX/CS nanocomplexes, with an increase in
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proportion of EX, the particle size was decreased. A similar particle size change was
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observed for EX/PEI nanocomplexes. Additionally, the zeta potential of three
187
nanocomplexes was decreased with an increase of proportion of EX, which is
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attributed to the excess presence of negatively charged carboxylic groups and sulfate
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groups on EX molecules. Furthermore, nanocomplexes with higher EX/polymer mass
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ratio (EX/Plys ≥ 4:4; EX/CS ≥ 4:4; EX/PEI ≥ 4:2) became unstable and tended to
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precipitate, which was probably due to the excess EX could not bind polymers tightly
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at a certain amount of polymers. Meanwhile, in order to prepare stable, homogenous
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and concentrated nanocomplexes, EX/Plys = 3:4, EX/CS = 3:4, and EX/PEI = 3:2
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were chosen as optimal mass ratio. Consequently, these nanocomplexes were used to
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fabricate the thermoreversible hydrogels.
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The three optimal nanocomplexes were visualized by TEM. The TEM images of
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nanocomplexes are shown in Fig.2, indicating that all nanocomplexes were spherical,
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and the sizes were similar to the results obtained by dynamic light scattering
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technique. In addition, the encapsulation efficiency of three optimal nanocomplexes
200
was determined according to ultrafiltration method, and the EE for EX/Plys
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nanocomplexes, EX/CS nanocomplexes and EX/PEI nanocomplexes was 97.2%,
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98.5% and 98.2%, respectively, indicating that EX could almost completely bind
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these positively charged polymers.
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3.2 Optimization of concentration of F127
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Gelation temperature is the temperature at which the liquid phase undergoes the
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transition from solution to gel. Gelation temperature was determined according to the
ACCEPTED MANUSCRIPT above mentioned visual method. Gelation temperatures for plain F127 hydrogels were
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measured for the concentration range of 15%-24% (F1-F4). As shown in Table 2, it
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was found that gelation temperature of F127 hydrogels decreased with an increase of
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concentration of F127. In order to ensure that F127 hydrogels combined the
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advantages of convenient administration and long-acting drug depot, the prepared
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F127 hydogels should be liquid state at room temperature (25 ℃) and form semi-solid
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gels at body temperature (37 ℃). Hence, 21% (w/v) concentration of F127 was
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selected as for further studies. When drug solutions or nanocomplexes were added
215
into F127 hydrogels (F5-F8), it was found that gelation temperature of formulations
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did not change, which suggested that incorporation of drug could not cause
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modification of the process of micellar association of F127 hydrogels.
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F127 copolymer blocks based on poly (ethylene oxide) - b - poly (propylene
219
oxide) - poly (ethylene oxide) (PEO-PPO-PEO) sequences. Fig.3 illustrates the
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establishment of nanocomplexes in hydrogels and mechanism of gelation of F127.
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Namely, F127 forms micelles above critical micelles concentration (about 1 mg/ml)
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[26]. Further, below a lower critical solution temperature (< LCST), both ethylene and
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propylene oxide blocks are hydrated, and PPO is relatively soluble in water. As the
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temperature increases (> LCST), the polymer solution turns into a gel owing to the
225
micelles packing and entanglements.
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3.3 Scanning electron microscope (SEM) of thermo-sensitive hydrogels
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SEM micrographs of lyophilized hydrogels structure were presented in Fig.4.
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From the micrographs, we can see that the structures of four thermo-sensitive
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hydrogels were almost the same, and the inner structures were porous with mesh size
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of about 1 µm. This probably could be attributed to the same F127 concentration for
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those hydrogels.
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3.4 Dissolution of hydrogels
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In general, F127 hydrogels undergo dissolution in an aqueous environment
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owing to the water penetration into gel network, leading to unpacking of the F127
235
micelles, polymer hydration and finally hydrogels dissolution. The dissolution
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profiles of different F127 hydrogels are shown in Fig.5. From the above results, it can
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be concluded that hydrogels with EX solutions and EX nanocomplexes (EX/Plys
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nanocomplexes, EX/CS nanocomplexes, EX/PEI nanocomplexes) had similar
239
dissolution profiles for the same concentration of F127.
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3.5 In vitro release of EX Because these hydrogels were intended for subcutaneous administration,
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membraneless method was utilized to evaluate the in vitro release of EX from F127 in
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situ hygrogels, which is to be closer to the in vivo condition. Fig. 6 indicates that
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27.78%, 13.01%, 13.98% and 9.02% of EX were cumulatively released from solution
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hydrogels, EX/Plys nanocomplex hydrogels, EX/CS nanocomplex hydrogels and
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EX/PEI nanocomplex hydrogels at 20 h, respectively. In contrast with EX solution
247
hydrogels, there was a prolonged release of EX from the nanocomplexes hydrogels.
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Approximately 99.37% of the cumulative amount of EX was released from EX
249
solution hydrogels within 144 h. In the case of EX/Plys nanocomplexes hydrogels,
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EX/CS nanocomplex hydrogels and EX/PEI nanocomplex hydrogels, the percent
251
cumulative release of EX was only about 30.39%, 39.52% and 22.18% within 144 h.
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From above mentioned results, it can be concluded that EX nanocomplexes could
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prolong the release of EX from hydrogels.
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Three different release models were used to predict the drug release, and the
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regression results for the release of EX from hydrogels are shown in Table 3. The in
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vitro release of EX from solution hydrogels, EX/Plys nanocomplex hydrogels, EX/CS
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nanocomplex hydrogels and EX/PEI nanocomplex hydrogels corresponds to the
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Ritger-Peppas model with r values of 0.997, 0.983, 0.994, 0.991. Ritger-Peppas
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equation has three different meanings. One is in the case of n (release exponent) <
260
0.45, indicating that drug release is diffusion-controlled, namely Fickian diffusion.
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Yet another is in case of n > 0.89, indicating that drug release is erosion-controlled.
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Values of n between 0.45 and 0.89 can be considered as an indicator for diffusion-
263
and erosion- controlled dual release mechanism. From the Table 3, it can be
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concluded that drug released from solution hydrogels or nanocomplex hydrogels was
265
controlled by diffusion and erosion. As we know, erosion is determined by F127
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hydrogels dissolution, and F127 hydrogels dissolution mainly depends on F127
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depends on the mesh sizes within the hydrogels matrix, but also on hydrodynamic
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radius of the drug molecules [27]. In our study, the mesh sizes almost were the same
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(Fig.4) due to the same concentration of F127 and preparation method. The size of EX
271
solution and three nanocomplexes was smaller than 1 µm (hydrogels pore size)
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Therefore, for EX solution hydrogels, EX could diffuse from hydrogels freely along a
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concentration gradient. In addition, some EX would release while hydrogels
274
dissolution. For EX nanocomplexes hydrogels, the release process was relatively
275
complicated. Maybe three kind of processes co-exist. First, EX nanocomplexes also
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could diffuse from pores of hydrogels, and then EX released from nanocomplexes
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through nanocomplexes dissociation. Second, EX dissociated from nanocomplexes
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and then released from the hydrogels. Third, EX directly released from hydrogels with
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hydrogels dissolution. The first two may be the main reason why nanocomplexes
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prolong the release of EX in hydrogels. Besides, among three nanocomplexes, EX/CS
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nanocomplexes release rate was relatively faster than other two nanocomplexes.
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During the course of experiment, the pH of the EX/CS nanocomplexes system was
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gradually increasing after the addition of the fresh release medium (pH 7.4). Chitosan
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molecules deprotonated and precipitated, resulting in faster dissociation of
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nanocomplex and EX release due to chitosan deionization. For EX/Plys
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nanocomplexes and EX/PEI nanocomplexes, they keep stable in release medium, so
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EX released from the two groups was relatively slower. Also, EX released from
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EX/PEI nanocomplexes was the slowest, this probably was due to the strong
289
electrostatic interaction between EX and PEI polymer, which has rather high charge
290
density. From above all, it can be concluded that the combination of thermo-sensitive
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hydrogels and nanocomplexes could significantly sustain the drug release, and the
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drug release from nanocomplexes/hydrogels was controlled by hydrogels dissolution
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and nanocomplexes dissociation.
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3.6 Correlation between hydrogels dissolution and drug release
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To further investigate whether the difference observed in EX release between
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solution/hydrogels and nanocomplexes/hydrogels is due to the hydrogels dissolution
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and nanocomplexes dissociation, the correlation between the percentage of EX
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released and the percentage of hydrogels dissolved was linear fitted. As shown in
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Fig.7, it can be seen that good linear correlation between them, which indicated that
300
theses
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Additionally, the linear order was solution (R² =0.9952) > EX/CS nanocomplex (R²
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=0.9867) > EX/Plys nanocomplex (R² =0.9720) > EX/PEI nanocomplex (R² =0.9636),
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which
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dissociation-controlled. Therefore, EX released from nanocomplex/hydrogels is both
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hydrogels dissolution- and nanocomplexes dissociation-controlled.
primarily
demonstrated
that
hydrogels
EX
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released
release.
from
nanocomplex
also
4. Conclusion
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dissolution-controlled
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are
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formulations
In this study, we developed an EX nanocomplexes/F127 thermo-sensitive
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hydrogel composite system which possessed the same thermo-sensitive property as
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blank F127 hydrogels, and achieved prolonged drug release. Three positively-charged
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polymers were used to prepare EX polyelectrolyte nanocomplexes. In vitro release
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results indicated that nanocomplexes fabricated by different polymers held different
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release rates, and PEI polymer could preferably sustain the EX release. In order to
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clarify how the PEI affected the drug release from the hydrogel systems in detail,
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different concentration and molecular weight of PEI will be investigated in our study
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in future.
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Acknowledgements
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This work was supported by the Natural Science Fund for Colleges and Universities
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in Jiangsu Province (No. 17KJB350009) and Natural Science Foundation of Jiangsu
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Province (No. BK20170445).
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ACCEPTED MANUSCRIPT Table 1 Physicochemical characteristics of nanocomplexes at various EX/polymer mass ratios.
2:4 3:4 4:4 5:4
EX/CS
1:4 2:4 3:4
1:2
3:2
4:2
194.93±3.11
0.109±0.025
32.38±1.67
143.70±4.13
0.143±0.033
184.53±2.64
0.110±0.008
804.36±72.35
0.141±0.104
588.72±19.37
0.218±0.022
59.12±1.95
519.31±1.36
0.241±0.032
57.00±1.28
0.240±0.017
52.76±1.40
0.282±0.035
48.84±1.16
302.27±4.85
0.207±0.038
47.31±2.15
1082.54±40.01
0.377±0.043
7.70±0.77
442.20±79.73
0.325±0.039
4.69±2.75
319.91±6.10
0.172±0.049
-14.59±1.73
146.32±2.23
0.173±0.029
-22.94±0.79
114.11±0.687
0.272±0.007
-38.82±5.13
325.14±12.49
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39.96±0.30
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0.5:2
0.357±0.056
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EX/PEI
1091.06±469.36
386.61±0.60
4:4 5:4
Zeta potential (mV)
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Polydispersity index
27.96±2.53
23.43±2.21
-26.91±2.21
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EX/Plys
Size (nm)
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Table 2 Results of optimization of concentration of F127. Formulation batch
F1
F2
EX (mg/ml) Polymer (mg/ml) F127(%,w/v) Tsol-gel ( )
0 0 15
0 0 18 37
﹥60
F3
F4
F5*
F6*
F7*
F8*
0 0 21 28
0 0 24 22
0.6 0 21 28
0.6 0.4 21 28
0.6 0.4 21 28
0.6 0.4 21 28
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Tsol-gel, gelation temperature;
*, F5-F8 respectively represents EX solution hydrogels, EX/Plys, EX/CS, EX/PEI nanocomplexes
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Table 3
Release kinetics of EX solutions, EX/Plys nanocomplexes, EX/CS nanocomplex and EX/PEI nanocomplex hydrogels.
Zero order
First order
Ritger-Peppasa
Q versus t
ln (1 − Q) versus t
lnQ versus lnt
EX solutions hydrogels
y = 0.664x + 9.591
y = -0.664x + 90.409
y = 0.709x + 1.121
EX/Plys nanocomplexes hydrogels
r = 0.985 y = 0.180x + 5.888
r = 0.985 y = -0.180x + 94.113
r = 0.997 y = 0.517x + 0.859
EX/CS nanocomplex hydrogels
r = 0.947 y = 0.236x +6.120
r = 0.947 y = -0.236x + 93.880
r = 0.983 y = 0.540x + 0.947
r = 0.966 y = 0.137x +4.267 r = 0.952
r = 0.966 y = -0.137x + 95.733 r = 0.952
r = 0.994 y = 0.5201x + 0.553 r = 0.991
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Formulation
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Ritger-Peppas equation: lnQ = nlnt + lnk, Q is the fractional drug release, n is the release exponent, t is the release time and k is a rate constant.
ACCEPTED MANUSCRIPT Figure captions Fig.1. Structure of (a) enoxaparin, (b) polylysine, (c) chitosan and (d) polyethyleneimine. Fig.2. TEM micrograph of (a) EX/Plys nanocomplexes, (b) EX/CS nanocomplexes
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and (c) EX/PEI nanocomplexes. Fig.3. Schematic structure of nanocomplex in F127 hydrogels. LCST: low critical solution temperature.
Fig.4. SEM micrograph of (a) EX solution in hydrogels, (b) EX/Plys nanocomplexes
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in hydrogels, (c) EX/CS nanocomplexes in hydrogels and (d) EX/PEI nanocomplexes in hydrogels.
(Ph 7.4, 37
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Fig.5. Dissolution profiles of thermoreversible hydrogels at physiological conditions ). Data are means ±SD of three measurements.
Fig.6. Release profiles of EX from F127 hydrogels (21%). Data are means ±SD of three measurements.
Fig.7. A correlation of cumulative percent of EX released with cumulative percent of
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F127 hydrogels. The line represents a linear regression. (a) EX solution in hydrogels, (b) EX/Plys nanocomplexes in hydrogels, (c) EX/CS nanocomplexes in hydrogels and
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(d) EX/PEI nanocomplexes in hydrogels.
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polyethyleneimine.
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Fig.1. Structure of (a) enoxaparin, (b) polylysine, (c) chitosan and (d)
Fig.2. TEM micrograph of (a) EX/Plys nanocomplexes, (b) EX/CS nanocomplexes and (c) EX/PEI nanocomplexes.
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Fig.3. Schematic structure of nanocomplexes in F127 hydrogels. LCST: low critical
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solution temperature.
Fig.4. SEM micrograph of (a) EX solution in hydrogels, (b) EX/Plys nanocomplexes in hydrogels, (c) EX/CS nanocomplexes in hydrogels and (d) EX/PEI nanocomplexes in hydrogels.
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Fig.5. Dissolution profiles of thermoreversible hydrogels at physiological conditions ). Data are means ±SD of three measurements.
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Fig.6. Release profiles of EX from F127 hydrogels (21%). Data are means ±SD of three measurements.
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Fig.7. A correlation of cumulative percent of EX released with cumulative percent of F127 hydrogels. The line represents a linear regression. (a) EX solution in hydrogels,
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(b) EX/Plys nanocomplexes in hydrogels, (c) EX/CS nanocomplexes in hydrogels and
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(d) EX/PEI nanocomplexes in hydrogels.