Injectable alginate microsphere/PLGA–PEG–PLGA composite hydrogels for sustained drug release

Abstract

Microspheres and in situ formed hydrogels are widely used in drug delivery systems due to their excellent biocompatibility and convenient administration. However, both of them often show burst-release behavior. To overcome this disadvantage, a new composite system combining the advantages of microspheres and injectable hydrogels was prepared via mixing drug-loaded alginate microspheres with poly(D,L-lactide-co-glycolide-b-ethylene glycol-b-D,L-lactide-co-glycolide) (PLGA–PEG–PLGA) hydrogels without using any surfactant or organic solvent. Alginate microspheres with tunable size were prepared by an electrospinning method. The structure and surface morphology of microspheres were observed by optical microscopy and scanning electron microscopy (SEM), and the rheological properties of the PLGA–PEG–PLGA hydrogels were evaluated using a rheometer. 5-Fluorouracil (5-Fu) and theophylline (TP) were chosen as model drugs, and were encapsulated in the composite hydrogels. The drug release results demonstrate that this combined system could apparently solve the burst-release problem of both hydrogels and microspheres and achieve a sustained release through the double barriers by greatly reducing the diffusion of water-soluble drugs. These results indicate that the double-barrier composite systems have great potential application in sustained release systems.

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1 Introduction

Over the past several years, controlled drug delivery systems (DDS) have attracted great attention. Compared to the classical concept: a vehicle to deliver the drug into a biological system,¹ the new delivery system needs a more prolonged and a better control of drug administration, meanwhile retaining the bioavailability of the drugs.^2,3 Hydrogels, micro-/nanospheres, and micelles are widely used as drug carriers.^4–8 Among them, injectable hydrogels prepared with physical thermogelling are especially attractive due to their smart-responsive properties.⁹ For example, temperature-/pH-sensitive polymers could transform into an insoluble gel at body temperature or particular pH value, thus forming a controlled delivery vehicle for drugs.^10–12 However, an initial burst release is often observed because of its swelling properties.¹³

In recent years, microparticles have been increasingly studied as drug delivery vehicles for sustained drug release.^14,15 With good biocompatibility and degradability, alginate microspheres have attracted many attentions in drug delivery systems.^16–18 The production of alginate microspheres is traditionally achieved by injecting alginate solution into calcium chloride solution, which is a relatively fast and mild gelation process.¹⁹ Unfortunately, this method lacks control over the particle size and size distribution. Microspheres with diameter less than 100 μm can be obtained using a water-in-oil (W/O) emulsion technique.^20–22 However, the surfactants employed to stabilize the microspheres can not be easily cleared away and they usually have adverse effect on drug quantification. Electrospinning was often used to produce nanofibers as scaffolds in tissue engineering. The fiber's morphology and diameter could be controlled by changing the processing parameters.^23,24 However, rare attention has been paid to prepare microspheres by electrospinning in aqueous phase. In this study, we used electrospinning to produce more controllable alginate microspheres without using any surfactant and organic solvent.

Alginate (ALG) microspheres have been deeply explored as injectable delivery systems for sustained release. However, two serious problems still exist. Firstly, microspheres would cause rejection in vivo and would be shortly cleared by phagocytes after administration. Secondly, the microspheres often show burst-release behavior in phosphate buffered saline (PBS) due to the unstable nature of ALG, especially when hydrophilic drugs are entrapped.^25,26 Many studies have been carried out to solve these problems with chemical and physical modifications. Chitosan-treated ALG beads have been developed as an attempt to suppress drug diffusion and erosion of gel matrix.²⁷ Pietro Matricardi prepared ALG microspheres interacted with a new copolymer poly-[(3-acrylamidopropyl)-trimethylammonium chloride-b-N-isopropylacrylamide], the release behavior of model molecules greatly depended on the temperature and enzyme activity, and a reduction in the burst release was observed.²⁸ In another research, ionic cross-linked hydroxamated alginic acid suppressed the swelling of microspheres and has proved to be successful in decreasing the burst release.¹⁵

Several composite systems have also been explored to control the drug or growth factor release.^29–33 Kacey G. Marra embedded poly(lactic-co-glycolic acid) (PLGA) microspheres in poly(ethylene glycol) (PEG)-based hydrogel, the composite had a more controlled release of TGF-β1 to cartilage wound sites.³⁴ Kuen Yong Lee prepared an injectable system with combining PLGA microspheres and alginate hydrogel for localized protein delivery.²⁶ However, the PLGA microspheres were usually prepared in organic phase in the presence of emulsifier. By employing a green way, the aim of this work is to develop injectable alginate microspheres/poly(DL-lactide-co-glycolide-b-ethylene glycol-b-DL-lactide-co-glycolide) (PLGA–PEG–PLGA) composite hydrogels as a double barrier system to reduce the burst release and to achieve a prolonged release system for hydrophilic drugs without using any organic solvent and surfactant. This new double-barrier system could congregate the advantages of these two materials and limit the burst release, while achieving a controlled and sustained release of water soluble drugs. The system was prepared through blending PLGA–PEG–PLGA hydrogel with alginate microspheres as the injectable delivery system, as Fig. 1 described. Alginate microspheres containing different water-soluble drugs were dispersed in PLGA–PEG–PLGA hydrogels and the release profiles were evaluated.

Fig. 1 Schematic description of an injectable microsphere-loaded hydrogel composite.

2 Materials and methods

2.1 Materials

Alginic acid sodium salt (ALG), calcium chloride, 5-fluorouracil (5-Fu), theophylline (TP) (99%) and poly(ethylene glycol) with a molecular weight of 1500 (PEG1500) were obtained from Sigma. Lactide (LA) and glycolide (GA) from Sigma were recrystalized before use. Stannous 2-ethylhexanoate (96%) was purchased from Alfa and was dried over molecular sieves.

2.2 Preparation of alginate microspheres by electrospinning

The electrospinning device used in this study was applied by UCALERY BEIJING CO., Ltd. A series of ALG solutions with different concentrations (0.5 wt%, 1 wt%, and 2 wt%) were placed into a 5 mL syringe attached to a syringe pump. Under controlled voltage and injection speed, the resulting microdroplets were converted into ALG microspheres by gelation in CaCl₂ solution (4 wt% or 8 wt%) through a No. 21 needle. The microspheres were collected by centrifugation at 4500 rpm for 5 min, and followed by washing with deionized water for 3 cycles.

2.3 Morphology of ALG microspheres

The morphology of moist microspheres was observed using an optical microscope (AxioCam MRc 5, zeiss), and the morphology of dried microspheres was observed by scanning electron microscopy (FE-SEM, SU-8000, Hitachi, Japan). The diameter of microspheres was evaluated from at least 100 measurements of microspheres in a randomly chosen area. The average diameter and standard deviation were reported.

2.4 Preparation of temperature-sensitive hydrogel

Temperature-sensitive copolymer PLGA–PEG–PLGA with a feed molar ratio of LA to GA = 6 : 1 was synthesized by a ring-opening polymerization.^35,36 Briefly, 4 g PEG1500, 8.464 g LA and 1.136 g GA dried under vacuum for 48 h were added in a 100 mL round bottom flask. 17.6 mg stannous 2-ethylhexanoate as a catalyst was added to the above flask in a glove box purged with nitrogen (MBraun lab star). The flask was then put in an oil bath at 120 °C for 48 h. The product was dissolved in cold water (4–8 °C) and purified by heating the system to 80 °C. After 2–3 times of dissolution and precipitation, the purified copolymer was vacuum-dried.

2.5 Hydrogel characterization

The structure and composition of the copolymer PLGA–PEG–PLGA was characterized by a Bruker Ascend 400 MHz NMR instrument, with CDCl₃ being used as solvent and internal standard. ¹H NMR (400 MHz, CDCl₃) δ 5.18 (t, 1H, poly –CH–), 4.80 (t, 2H, poly –CH₂–), 4.37 (t, 2H, end –CH₂– from PEG), 3.60 (m, 4H, –OCH₂CH₂–), 1.60 (d, 3H, poly –CH₃), 1.51 (d, 3H, end –CH₃).

The sol-to-gel transition temperature of PLGA–PEG–PLGA solution was determined by a TA rheometer (DHR-2). Polymer solution of different concentrations was prepared in deionized water and the gelation behavior was observed at temperature ranging from 0 °C to 60 °C. The experiment was carried out at a frequency of 1 Hz, 1.6% strain, and a 0.5 °C min⁻¹ temperature ramp with a parallel plate.

2.6 In vitro drug release test

5-Fu and TP were chosen as model drugs for the controlled release experiment. The microspheres obtained by electrospinning were dried in a 37 °C oven for 12 h. The microspheres were then dispersed in a dialysis bag (molecular weight cutoff 2000) and put into saturated solution of different drugs (5-Fu or TP) for 72 h under 100 rpm. Small molecular drugs were transferred into swollen microspheres through diffusion until balance.

Drug loading in microspheres was estimated in terms of the following formula:

Drug loading = W_d/W_a × 100%,

where W_d is the amount of drugs in the microspheres and W_a is the weight of ALG microspheres. The drug-loaded microspheres in dialysis bag were then placed into a flask containing 100 mL PBS buffer solution (pH 7.4). The flask was put into a constant temperature-shaking incubator at 37 °C with 100 rpm. At every predetermined time interval, 1 mL of the release media was taken out, meanwhile 1 mL fresh PBS was supplemented. The concentration of drugs in supernatant was quantified via ultraviolet spectrophotometer (PerkinElmer Lambda 35). The UV-vis absorption of 5-Fu and TP was 266 nm and 271 nm, respectively. For each sample, the experiments were repeated three times and the final results were calculated averagely.

5 mg 5-Fu or TP was dissolved in 1 g 15 wt% PLGA–PEG–PLGA solution. After the drugs were totally dissolved, the solution was placed in a 37 °C oven for 15 min to obtain the drug-loaded hydrogel.

The drug-loaded microspheres were physically blended with 15 wt% PLGA–PEG–PLGA aqueous solution and stirred for 10 min to guarantee the microspheres dispersed evenly in hydrogel. After the gelation of PLGA–PEG–PLGA solution, the drug-loaded ALG microsphere hydrogel composite was obtained.

The in vitro drug release from hydrogel and microspheres/hydrogel composites was carried out in a similar manner to the drug release from the microspheres.

3 Results and discussion

3.1 Fabrication of ALG microspheres by electrospinning

Typical parameters that influence diameter of microspheres in electrospinning include voltage, ALG concentration, calcium chloride concentration and injection speed. To optimize fabrication parameters for ALG microspheres, ALG microspheres with different formulations were produced. Table 1 shows the effect of formulation variables on the diameter of the ALG microspheres.

Table 1 Effect of formulation variables on the diameter of ALG microspheres

Sample	ALG concentration	CaCl2 concentration	Injection speed (mm min^−1)	Voltage (kV)	Diameter (μm)
I	1%	4%	0.08	8	187 ± 16
II	1%	4%	0.08	10	125 ± 5
III	1%	8%	0.08	8	152 ± 15
IV	1%	8%	0.08	10	108 ± 6
V	1%	8%	0.1	10	131 ± 8
VI	1%	8%	0.06	10	103 ± 11
VII	1%	8%	0.08	8	152 ± 15
VIII	1%	8%	0.08	9	139 ± 4
IX	1%	8%	0.08	10	108 ± 6
X	1%	8%	0.08	11	106 ± 5

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As shown in Table 1, changing voltage during electrospinning could well control the diameter of microspheres, that is, the size of the microsphere decreased obviously with voltage increasing from 8 kV to 10 kV. However, when using higher voltage than 10 kV, the Taylor cone caused by high voltage made microspheres with small tails or gaps, and with higher voltage, the microdroplet beam became unstable, resulting in uneven bigger microspheres.

The concentration of ALG and calcium chloride also deeply influenced the diameter of microspheres. When the concentration of ALG is 0.5% or 2%, the viscosity was too low or too high to carry out the electrospinning smoothly. On the other hand, the more concentrated calcium chloride solution, microspheres with the smaller size were obtained. This was because higher concentration of calcium chloride would restrain swelling properties of microspheres, thus producing smaller size of microspheres. In addition, we found that slower speed would produce smaller microspheres, but when the speed was lower than 0.06 mm min⁻¹, the micro-droplets were not stable and the decrease of the size of the microsphere was not obvious.

3.2 Morphological properties of microspheres

The morphology of ALG microspheres was observed by optical microscope and SEM. Fig. 2 shows representative optical images of ALG microspheres (samples I, II, III and IV in Table 1). It can be seen from Fig. 2 that all the ALG microspheres in wet state have a quite round shape, and exhibit a uniform distribution. Fig. 3 displays representative SEM images of the dried microspheres (samples I, II, III, and IV in Table 1). Generally, all the microspheres showed a spherical morphology with a condensed surface, and the diameters of microspheres shranked to about 30–85 μm due to the drying effect in oven. Sample I with the largest size in wet state still showed the bigger size around 85 μm than other samples (II, III and IV) after drying.

Fig. 2 Morphological properties of (a) sample I, (b) sample II, (c) sample III, and (d) sample IV by optical microscope.

Fig. 3 SEM images of ALG microspheres, (a) sample I with low magnification, (b) sample I with high magnification; (c) sample II with low magnification, (d) sample II with high magnification; (e) sample III with low magnification, (f) sample III with high magnification; (g) sample IV with low magnification, and (h) sample IV with high magnification.

By summarizing the fabrication parameters and morphology observation, we could achieve the optimal parameters for preparing ALG microspheres with good morphology as follows: the voltage is 8–10 kV, the concentration of calcium chloride is 4–8 wt%, the concentration of ALG is 1 wt%, and the injection speed is 0.08 mm min⁻¹. Therefore, we use these parameters to produce the microspheres for the drug release study in the following work. Since the sizes of the microspheres affect the drug loading and drug release behavior of the materials, we chose the microspheres with diameters of 108 μm, 125 μm, 152 μm and 187 μm (samples IV, II, III and IV in Table 1) for drug release test because these microspheres cover the whole size range of the samples.

3.3 Properties of hydrogel

The PLGA–PEG–PLGA copolymer was synthesized by the ring-opening polymerization. The PLGA–PEG–PLGA copolymer with a molecular weight of 5300 and a LA/GA molar ratio of 3 : 1 were confirmed by NMR spectra. The rheological property of the PLGA–PEG–PLGA solution was studied by rheometer. Gelation temperature is defined as the temperature when storage modulus G′ is higher than loss modulus G′′. The sol-to-gel transition temperature of 15 wt% PLGA–PEG–PLGA solution was found to be at 36 °C, as shown in Fig. 4. At about 38.5 °C, the storage modulus and complex viscosity of the hydrogels were both the highest, meaning that the hydrogel reached the highest strength. Therefore, the copolymer has a relatively higher strength at body temperature and would probably slow down the free diffusion of the water-soluble drugs.

Fig. 4 Sol-to-gel transition of the PLGA–PEG–PLGA copolymer solution.

3.4 In vitro drug release from microspheres

5-Fu and TP were chosen as model drugs for the controlled release experiment, both of which are hydrophilic and with small molecular weights. Table 2 showed the relationship between the size of microspheres and drug loading. It was found that the bigger particle size could improve the 5-Fu loading by comparing sample I with II and sample III with IV. This could be because the bigger microsphere would swell larger, which provided more space for the encapsulation of drugs. By comparing the samples I, II with III, IV, we can see that the higher calcium chloride concentration would reduce the drug loading, probably because the more concentrated calcium chloride would restrain the swelling of microspheres, thus resulting in a lower 5-Fu loading.

Table 2 Effects of microsphere size on drug loading of 5-Fu and TP

Sample	Diameter (μm)	CaCl2 concentration	Drug loading of 5-Fu	Drug loading of TP
I	187 ± 16	4%	54.8%	25.4%
II	125 ± 5	4%	35.8%	19.6%
III	152 ± 15	8%	35.3%	18.5%
IV	108 ± 6	8%	19.4%	16.7%

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The cumulative release of these two drugs from the ALG microspheres was showed in Fig. 5. Both of two drugs showed an obvious burst release from ALG microspheres. Some of them even exhibited a burst release of 80% in half an hour. This could be attributed to the release of drug adhered on the surface of the microspheres, and good solubility of the small molecular drugs. Most of the system released 80–90% of the drug encapsulated in the microspheres at 2 h due to the free diffusion of the drug molecules. Drug release rates can be quantified in terms of drug release half-time of the bioactive agent from a carrier. The term “half-time” means the time needed to release 50% of the initial drug payload encapsulated in the microspheres. The drug release half-times for 5-Fu and TP were 12 min and 12–17 min, respectively, which are quite short.

Fig. 5 In vitro release from ALG microspheres at pH 7.4 solution, (a) 5-Fu and (b) TP.

3.5 In vitro drug release from hydrogels

PLGA–PEG–PLGA is a water soluble polymer below the gel transition temperature, and it forms a water-insoluble gel rapidly once injected into human body. Because of its good biocompatibility, permeability and biodegradability, it has been widely used in drug delivery systems.^35,37,38 Fig. 6 shows the release profile of 5-Fu and TP from PLGA–PEG–PLGA hydrogel. It was found that about 60% of both drugs were released from the injectable hydrogel in 1 h, and over 80% of the drugs were released in 5 h. The hydrophilic drug would diffuse fast from the hydrogel matrix because the hydrogel swelled, and these results agreed with other reports.^13,39,40 The release half-times for 5-Fu and TP in the hydrogel system were 40 min and 30 min, respectively.

Fig. 6 Drug release from PLGA–PEG–PLGA hydrogels.

3.6 In vitro drug release from the microspheres/hydrogel composites

In this study, we used ALG microspheres composited with PLGA–PEG–PLGA hydrogel to form an injectable double-barrier system to prolong the hydrophilic drug release. Fig. 7 displays the kinetics of TP release from the composite system. We could see the 1 h TP release percentage was reduced to around 20%; meanwhile the total TP release time was prolonged to 24 h. The 5-Fu release showed a similar release result (data not shown). The release process could be divided into two steps: firstly, the initial released drugs were attributed to the drugs attached to the surface of ALG microspheres. In the second stage, the drugs encapsulated inside the microspheres had to permeate through two barriers, i.e. microsphere compartment and hydrogel structure. The double buffer barriers increased the opportunities of collision between the drugs, thus slowing down the diffusion speed of the drugs, which resulted in a prolonged release of the drug molecules out of the matrix. The composite systems showed drug release half-times between 136 min and 318 min depending on the different sizes of the ALG microspheres. This means that the composite system greatly increased the drug release half-time compared to the microspheres or hydrogels.

Fig. 7 TP release from the double-barrier systems.

We found that the different diameter of microspheres had certain influence on the release profiles. The sample I in Table 2, i.e. microspheres produced under 4% calcium chloride and 8 kV, showed a slowest release because of their biggest size. At the same time, with microspheres' size decreasing, the release speed became faster. A possible reason is that the bigger microspheres have more space, thus increasing the entrapment of the drugs and delaying the diffusion speed.

To clearly compare the release profiles from the ALG microspheres, PLGA–PEG–PLGA hydrogel and their composites, their release profiles were plotted together and the results were shown in Fig. 8. Apparently, the drug release from the ALG microspheres or hydrogel showed an obvious burst release, and more than 80% of the drug was released from the matrix in 1 h. However, the drug release percentage was only about 20% at 0.5 h, and it increased to 29% at 1 h and 40% for 2 h, indicating that the burst release of the microsphere/hydrogel composite was greatly reduced. The drug was released gradually after the first 2 h, and the release percentage was about 80% at about 20 h. Therefore, this double-barrier system can prolong the release of small molecular hydrophilic drugs, like 5-Fu and TP by greatly suppressing the initial abrupt release, meaning that a constant drug dose for 1 day can be easily achieved when the microsphere/hydrogel composite was used as drug carrier.

Fig. 8 Comparative release profiles of these three type systems, left: TP, right: 5-Fu.

4. Conclusions

We demonstrated injectable composite hydrogels composed of alginate microspheres and PLGA–PEG–PLGA hydrogel as a double barrier system to greatly suppress the burst release and to prolong the release time of two small molecular water soluble anti-inflammatory drugs. The alginate microspheres were prepared by electrospinning without any surfactant and the composite system of alginate microspheres and PLGA–PEG–PLGA hydrogel was fabricated in a green manner which was free of any organic solvent. The burst release of TP from alginate microspheres or hydrogel alone was between 39–78% for 30 min, and the double barrier system decreased the burst release to 17% for 30 min. The two barrier matrix prolonged the release time 4–6 times compared to the alginate microspheres or hydrogel alone system. The new double barrier system prepared with a green manner has a great potential application for new drug delivery systems.

Acknowledgements

The authors gratefully acknowledge the Natural Science Foundation of China (grant number 21304073) and Xi'an Jiaotong Univerisity for financial support of this work.

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