Production and characterization of hyaluronic acid microparticles for the controlled delivery of growth factors using a spray/dehydration method

Abstract

Hyaluronic acid is the main polysaccharide present in the connective tissue. Besides its structural function as backbone of the extracellular matrix, hyaluronic acid plays staple roles in several biological processes including the modulation of inflammation and wound healing processes. The application of hyaluronic acid in regenerative medicine, either as cells and/or drug/growth factors delivery vehicles, relies on its ability to be cross-linked using a plethora of reactions, producing stable hydrogels. In this work, we propose a novel method for the production of hyaluronic acid microparticles that presents several advantages over others that have been used. Basically, droplets of hyaluronic acid solution produced with a nozzle are collected in an isopropanol dehydration bath, and stabilized after crosslinking with adipic acid dihydrazide, using a cabodiimide-based chemistry. The size and morphology of the hyaluronic acid microparticles produced by this method varied with the molecular weight and concentration of the hyaluronic acid solution, the nozzle chamber pressure, the distance between the nozzle and the crosslinking solution, and the number of crosslinking steps. The degree of crosslinking of the hyaluronic acid microparticles produced was tunable and allowed to control the rate of the degradation promoted by hyaluronidase. Moreover, the particles were loaded with platelet lysate, a hemoderivative rich in cytokines with interest for regenerative medicine applications. The hyaluronic acid microparticles showed potential to bind selectively to positively charged molecules, as the factors present in the platelet lysate. It is envisioned that these can be further released in a sustained manner by ion exchange or by the degradation of the hyaluronic acid microparticles matrix promoted by extracellular matrix remodeling.

Keywords

Hyaluronic acid microparticles drug delivery system platelet lysate

Introduction

The hyaluronic acid (HA), also known as hyaluronan or hyaluronate,¹ is a high molecular mass linear polysaccharide of alternating d-glucuronic acid and N-acetyl-d-glucosamine.^2,3 The molecular weight typically ranges from 10⁶ to 10⁷ Daltons.⁴

First isolated from the vitreous humor of bovine eyes by Meyer and Palmer in 1934,⁵ the HA is a core component of extracellular matrix (ECM). It is present in high concentrations in most of typical connective tissues, namely the umbilical cord, the synovial fluid, or in the fibrous matrix of skin or vitreous humor.⁶

The hyaluronan interacts with cells via three main classes of cell surface receptors: CD44, ICAM-1, and RHAMM (receptor for Hyaluronan mediated motility expressed protein).^7,8 HA is implied in the modulation of inflammation⁹ and wound healing processes, including angiogenesis and endothelial cell migration¹⁰ and morphogenesis.¹¹

HA is naturally degraded by hyaluronidases (HAses)¹² which hydrolyze the hexosaminidic β(1 → 4) linkages between N-acetyl d-glucosamine and d-glucuronic acid residues in HA. The HAses are present in human testis, kidney, urine, plasma, synovial fluid, and synovial tissue. The degradation rate of HA mediated by HAses can be increased in case of injury.³ Thus, the HA hydrogels have the advantage to be degraded and release their content in a requested manner.

An important feature of HA applicability in regenerative medicine applications is the ability of HA to be readily modified through both its carboxyl^13–16 and hydroxyl^17,18 groups. The crosslinkage of the carboxyl or hydroxyl groups of the glucuronic acid moieties has being exploited to produce stable hydrogels, or beads that can be used for various different applications, such as for cell encapsulation/delivery or as vehicles for controlled release of drugs, genes, or growth factors.^{14–16,19,20} Several methods have been proposed to obtain HA beads or microparticles, namely solvent evaporation and spray-drying methods,²¹ water in oil emulsion,¹⁵ and gas anti-solvent (GAS) precipitation.²⁰

In the present work, we propose a novel method for the production of biodegradable HA beads to be used as growth factors/drug delivery applications in regenerative medicine. This method consists of the injection of a solution of HA and adipic acid dihydrazide (ADH) through a nozzle to produce a beam of particles that are collected in a solution containing carbodiimide, which promotes the crosslinking of the particles by a reaction previously described for the production of HA hydrogels and microparticles.^14,15 This method holds several advantages over previously reported methods. The above-mentioned reaction occurs at room temperature (instead of using the high temperatures employed in the solvent evaporation and spray-drying methods²¹ that can thermally denature thermosensible compounds, such as proteins) and allows the usage of native HA. In addition, the chemistry used is non-toxic and the reaction by-products of the crosslinking such as urea and unreacted reagents can be easily removed by conventional methods such as dialysis, precipitation, and ultra-filtration.¹⁵ Moreover, since the particles are produced without the use of surfactants and mineral oil, as in the water in oil emulsions,^15,21 the process in less toxic and pollutant.

In order to assess the potential of the microparticles produced by this method for the incorporation and delivery of growth factors, these were loaded with platelets lysate (PL) and the elution of proteins through time evaluated. PL constitutes a natural source of GFs including platelet-derived growth factor (PDGF), transforming growth factors (TGF)-β1 and -β2, insulin-like growth factor (IGF), epidermal growth factor (EGF), epithelial cell growth factor (ECGF), hepatocyte growth factor (HGF), bone morphogenetic proteins (BMPs)-2, -4, and -6, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF).^22–25 These GFs are involved in essential stages of wound healing and regenerative processes such as chemotaxis,²⁶ cell proliferation,²⁷ and differentiation, including angiogenesis,^22,25 chondrogenesis,²⁸ and osteogenesis.^29–31 Nevertheless, the GFs have a short lifespan, and lose their activity very fast. The incorporation of GFs into the polymeric matrix of the HA microparticles scaffolds is expected to extend their activity.³²

By combining the properties of HA microparticles as carriers for the delivery of macromolecules, and the biological relevance of the GFs contained in PL, we intend to develop a biodegradable delivery system with enhanced potential for promoting wound healing that can find numerous applications in regenerative medicine.

Materials and methods

Materials

Hyaluronic acid (HA) sodium salt from Streptococcus equi (Mws 66, 752, 830 and 2000 kDa) was purchased from Lifecore (Lifecore Biomedical, Chaska, USA). ADH ≥ 98%, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride – purum, ≥98.0% (EDCI), pyridine anhydrous, 99.8%, 5% methylene blue (MB) solution, hyaluronidase type IV from bovine origin (HAase), and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (USA). Sodium hydroxide (NaOH) and hydrochloride acid (HCl) were purchased from VWR Chemicals (BDH, Prolabo – international, USA). Formalin (10%) was purchased from BIO OPTICA (BIO OPTICA, Milan, Italy). All the other chemical reagents used were of analytical grade.

Preparation of platelet lysate

Platelet concentrates were provided by Instituto Português do Sangue (IPS, Porto, Portugal), under a previously established cooperation protocol and approved by the ethical committee of the respective institution. All the products were biologically qualified according to Portuguese legislation (Decreto-Lei n.º 100/2011). Platelet concentrates were processed as previously described,^33–35 to obtain PL. Briefly, platelet concentrate samples (with an average platelet count of 10⁶/µL) from at least three different donors were pooled and subjected to three repeated freezing and melting cycles (frozen with liquid nitrogen at −196℃ and thawed in a 37℃ water bath), thus lysing the platelets and releasing their protein content. The resulting cellular debris was removed by centrifugation at 1400 g for 10 min and the supernatant was stored at −20℃ until further use.

Production of hydrazide-crosslinked hyaluronic acid (HA) microparticles by spray/dehydration

Hyaluronic acid microparticles were produced using a spray/dehydration method, as depicted in Figure 1, followed by crosslinking with ADH, using a previously stablished cabodiimide-based chemistry.^16,36,37 Briefly, a HA/ADH solution was prepared by dissolving 1:1 weight ratio of HA and ADH (for concentrations ranging between 0.5 and 2 wt% of HA) in 100 mL of didistiled water (ddH₂O). The pH of the HA/ADH solution was set to 4.75 with 3M HCl and filtered. The HA/ADH solution was injected through the nozzle, with a nominal needle opening of 0.35 mm, of a Nisco Encapsulation Units VAR J30 (Nisco Engineering AG, Zurich, Switzerland) with a syringe pump. The pressure inside of the nozzle chamber was set by means of controlled compressed air injection, creating a beam of particles. These particles were precipitated in an isopropanol (IPA) solution, containing 2.5 molar excess of EDCI, and buffered with 1 mL of a 0.4 M pyridine solution (pH 4.75).

The particles were further separated from the crosslinking solution by centrifugation, and washed three times with ddH₂O, by centrifugation (1500 r/min, 5 min.).

A further crosslinking step, adapted from the method proposed by Yun et al.,¹⁵ was performed for some batches to increase the stability of the particles. In this case, the pellet of HA particles was re-suspended in a solution containing 0.6 g of ADH and 0.6 g of EDCI dissolved in 100 mL of 90% (v/v) IPA (in ddH₂O), at pH 4.0, and the reaction occurred overnight, under agitation.

The particles were further collected by centrifugation and washed three times with ddH₂O to remove the by-products and reagents remaining. Then, the HA microparticles were re-suspended in ddH₂O by ultrasounds, frozen at −196℃ in liquid nitrogen and freeze-dried.

In this study, several conditions/parameters of the HA microparticles production method were studied and optimized, namely the HA molecular weight, the concentration of the HA solution, the injection rate of the HA solution in the nozzle, the pressure inside of the nozzle chamber, the distance of the nozzle to the crosslinking solution, and the number of crosslinking steps. Table 1 summarizes the various sets of parameters used to produce different batches of HA microparticles.

Table 1.

Variables investigated for the optimization of HA microparticles production.

HA molecular weight (kDa)	HA concentration (%)	Sol. Rate (ml/min.)	Pressure inside nozzle chamber (barg)	Distance to the nozzle (cm)	CL cycles
66	0.50	0.25	0.3	10	1
752	0.75	0.50	0.4	15	2
830	1.00	0.75	0.5	19
2000	1.50	1.00
	2.00
	3.00

Characterization of the produced HA microparticles

Microparticles morphology and size

The morphology of the developed HA microparticles was examined by optical microscopy and scanning electron microscopy (SEM).

Freeze-dried samples from all the produced batches were dispersed in ddH₂O and the morphology of the HA particles observed under optical microscopy (Axio Imager Z1m, Zeiss). The particle diameter was assessed using the ImageJ software measuring tools.

For SEM analyses, the freeze-dried samples were sputter coated with gold and observed under a SEM (Leica Cambridge S360).

Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy (IR-Prestige-21, Shimadzu) was used to analyze the microparticles crosslinkage. For this purpose, the produced HA microparticles were mixed with potassium bromide and processed into pellets. The spectra were obtained in the range of 400–4000 cm⁻¹ at a 4 cm⁻¹ resolution with 32 scans.

Crosslinking density measurement

Methylene Blue (MB) is a cationic molecule with a high affinity to negatively charged solids, and therefore the absorption of MB in the HA microparticles can be related to the amount of ionic carboxylic acid. The MB sorption assay has been used for the quantification of carboxyl groups in cellulose³⁸ and ion capacity exchange in clay minerals.³⁹

In brief, dry HA microparticles were placed in 1.5 mL eppendorf tubes and weighted. A volume of 0.5% of Methylene Blue (MB) solution in ddH₂O (1.53 × 10⁻⁴ M) was added to the particles that were vigorously suspended in the MB solution by vortexing. The MB was allowed to adsorb to the particles for 1 h or until no color variation was noticeable. Subsequently, the supernatant was collected after centrifugation and fresh MB solution was added to the particles and the process was repeated. The absorbance of the supernatant was measured by spectrophotometry (Synergy HT, Bio-Tek Instruments, Winooski, USA) at λ=665 nm until no variation in methylene blue absorbance was observed. The concentration of MB in the solution was determined by comparison with a standard calibration curve.

The endpoint of the titration, or the point of complete cation replacement, was defined as the point for which the adsorption of MB was no longer 100%.³⁹ It was determined by the interception between the tangent line to the 100% of MB adsorption and the tangent to the slope of the adsorption efficiency decrease. The amount (in mol) of MB added until the endpoint was equivalent to the number of ionized carboxylic acid available in the HA microparticles sample.³⁹ Thus, the degree of crosslinking (D_CL) was calculated according to equation (1)

D CL = 1 - (n MB / 2) / (n ADH : HA) \times 100 %

(1)

where nMB is the amount of MB in mol, equivalent to the carboxylic moieties available in the HA microparticles sample, and the nADH:HA is the quantity in mol of polymer monomers in the known weight of HA microparticles.

Hyaluronic acid microparticles degradability

The degradation behavior of the particles was studied by immersion in a Hyaluronidase (HAase) solution. For this purpose, 4 mg of HA-ADH microparticles of formulations produced with different molecular weights (66 kDa, 830 kDa and 2 MDa) were suspended both in 400 µL of PBS and in 400 µL of Hyaluronidase solution (1000 U/mL in PBS, pH 6.3). The suspensions were incubated in a thermostatic water bath (OLS200, Grant) at 37℃, with agitation (60 r/min). Aliquots of 350 µL of medium were collected after centrifugation of the suspensions (5000 r/min for 5min.), upon 1, 3, 7 and 14 days of incubation, and replaced with freshly prepared solutions in the same volume. The collected aliquots were stored at −20℃ for further analysis. The degradation rate was measured as a function of N-acetyl-glucosamine (NAG) release, quantified using a colourimetric assay based on the Morgan–Elson reaction⁴⁰ modified by Reissig et al.⁴¹

Hyaluronic acid microparticles swelling

Swelling assays were conducted on 752 kDa HA microparticles. In a 1.5 ml eppendorff tube were added 4 mg of particles and the weight of the tube and the particles was registered. One milliliter of ddH₂O or PL was added to the particles. At pre-determined times, the supernatant was discarded by centrifugation (3000 r/min, for 3 min), and the pellet was gently blotted with filter paper and weighted. This procedure was repeated until the weight was stable.

Evaluation of hyaluronic acid microparticles ability for controlled release of PL

In order to assess the ability of the HA microparticles to uptake and release PL proteins, HA microparticles were suspended in PL and allowed to uptake the PL proteins for 15, 30, 45, and 60 min at room temperature. After each time frame, the particles' suspension was equally distributed for three 1.5 mL Eppendorf tubes (three replica), and the supernatant discarded by centrifugation (3000 r/min, for 3 min). Then, the microparticles pellet (corresponding to 6 mg of dry HA microparticles) was re-suspended in 1.5 mL of PBS and incubated at 37℃, pH 7.4, under a 60 r/min constant agitation, for a period of 24 h. At pre-determined time points (from 45 min to 24 h of incubation), aliquots of 0.4 mL were collected and stored at −20℃ and an equal volume of fresh PBS was added to the suspension to replace the sample. The amount of released protein in the supernatant was quantified using a total protein assay (micro-BCA, Thermo Fisher Scientific, USA), following the manufacturer's instructions. The absorbance was read at 562 nm on a multiwell microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, USA).

To determine the mechanism of PL protein release, the experimental release profile was fitted to the semi-empirical model developed by Korsmeyer et al.⁴² The release data up to the plateau of percent of protein release were used to produce theoretical release curves. Korsmeyer–Peppas model was developed to specifically model the release of a drug molecule from a polymeric matrix, such as a hydrogel and is defined by equation (2)

{Mt / M}_{\infty} = k_{KP} t^{n}

(2)

where M_t/M_∞ is the fraction of drug released at time t, k_KP is the rate constant, and n is the release exponent.

Considering the strong burst effect observed, the release kinetics was also analyzed using the modification of the Korsmeyer's semi-empirical equation by Kim and Fassihi.⁴³ The modified equation is given by equation (3)

M_{t} / M_{\infty} = k (t - t_{lag})^{n} + b

(3)

where M_t/M_∞ is the fraction of drug released before the lag phase (t−t_lag), k is the rate constant, n is the release exponent, and b is the total fraction of protein released from the burst effect.

Statistical analysis

All the experiments were performed with at least three replicates. Results are expressed as mean ± standard deviation (SD). Statistical analyses were performed with GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA) using one-way analyses of variance (ANOVA) with a Tukey's multiple comparison post-test. Differences between the groups with p < 0.05 were considered to be statistically significant.

Results and discussion

Production of hydrazide-crosslinked HA microparticles by spray/dehydration

In this work, we proposed a novel system for the production of HA microparticles that consisted in the production of a beam of HA particles by a nozzle, followed by their precipitation in an isopropanol solution. Since the HA is poorly soluble in the isopropanol, the HA drops maintained stable while the HA matrix was crosslinked with the ADH using a well-established chemical reaction promoted by a carbodiimide,^15,16 schematized in Figure 2(a). An extra crosslinking reaction was shown to increase the stability of the particles.

The proposed method offers several advantages over previously described processes for the production of HA microparticles. The large amount of organic solvents necessary to remove the surfactants and mineral oil used in the water in oil emulsions^15,21 are avoided, since the particles are produced using a spraying device. Furthermore, the spray method described herein produces particles in a semi-continuous regime,^20,21 thus allowing the production of larger batches than the emulsion methodologies. Moreover, since the by-products of the crosslinking reaction are non-toxic and easily removed by conventional methods such as dialysis, precipitation, and ultra-filtration,¹⁵ this method is more likely to produce in vitro and in vivo biocompatible particles.

The crosslinking of the particles after 1 or 2 crosslinking cycles was analyzed by FTIR. In Figure 2(b) it is possible to observe the appearance of new peaks at 1710 cm⁻¹ and 1569 cm⁻¹ characteristic of the ADH, that are more pronounced with the increasing of crosslinking. Moreover, the intensity of the peaks corresponding to the ionized carboxylic group of the hyaluronic acid sodium salt (1611 cm⁻¹ and 1416 cm⁻¹) progressively reduced with the number of CL reactions, consumed in the crosslinking reaction.

Optimization of microparticle size and morphology

In order to optimize the microparticles size and shape, experiments were performed to investigate the influence of process variables, such as polymer–solvent mixture concentrations, polymer molecular weight, injection flow rates, nozzle diameters and pressures, and CL effect. The morphology of the HA particles produced by each set of parameter (different batches) was analyzed by SEM after freeze drying, or by optical microscopy upon dispersion in di-distilled water (ddH₂O). In general, all the particles produced exhibited a nearly spherical shape and rough surface (Figure 3(a)).

The spray/dehydration method allowed the production of particles more regular in size and shape than other similar methods for the production of HA microparticles, namely GAS.²⁰ The most important limitation found in the production of microparticles was the high viscosity of the HA solutions, that increased with the molecular weight, and with the concentration of the HA solution used. High viscosity solutions offered higher resistance to the injection throughout the nozzle, often clogging inside of the nozzle needle. As a result, only the low (66 kDa) and medium molecular weights tested (830 kDa and 752 kDa) allowed for testing of all the conditions summarized in Table 1. Therefore, the results herein reported refer mainly to the data collected for particles produced with the medium molecular weights. Still, whenever it was possible to test the variables being studied for the low and high Mw HA, the microparticles yielded showed the same trend.

The main variables affecting the particle size were found to be the molecular weight and the concentration of HA in solution, most likely as a consequence of the increased viscosity of the polymer solution. The largest particle median size of the produced batches tended to increase with the HA molecular weight (Table 2). The number of CL cycles also affected the particle size. The increasing in the CL of the particles resulted in smaller and more homogeneous particles (Figure 3(b)), notably more stiff and stable than the particles produced with a single CL process. Likewise, the higher the concentration of HA, the bigger the particles, as shown in Figure 3(b).

Table 2.

Extreme dimensions of HA microparticle batches produced with the different HA molecular weight (Mw) tested in this study.

		Diameter (µm)			Batch synthesis conditions
Mw	Relative particle size	Median	Min. NO	Max. NO	HA wt%	nozzle pressure (barg)	distance nozzle/CL bath (cm)	CL cycles
2 MDa^a	Largest	50.58	4.63	99.86	1.0	0.30	16.0	2
2 MDa^a	Smallest	26.11	10.22	44.10	0.5	0.30	16.0	2
830 kDa	Largest	55.74	5.88	126.26	2.0	0.40	17.5	2
830 kDa	Smallest	17.98	5.52	34.07	0.5	0.40	16.0	1
752 kDa^b	Largest	39.52	7.29	72.25	1.0	0.30	10.0	1
752 kDa^b	Smallest	21.85	4.86	55.04	1.0	0.40	10.0	1
66 kDa	Largest	11.69	3.94	28.59	2.0	0.30	17.0	2
66 kDa	Smallest	7.17	1.37	13.95	0.5	0.40	10.0	2

Note: The size distribution for the batches with the largest and smallest median diameter produced for each HA Mw is represented as the median, minimum non-outlayer (Min. NO) and maximum non-outlayer (Max. NO) calculated for N>300 particle diameter measurements. The main variables contributing for the particles dimensions, namely the weight percentage of HA in solution (HA wt%), the gauge pressure (barg) inside the nozzle chamber, the distance of the nozzle to the crosslinking (CL) bath, and the number of CL cycles, are also indicated.

Given the high viscosity of the 2 MDa HA solutions, only the HA wt% and number of CL cycles were assessed.

Only the effect of nozzle pressure was assessed for 752 kDa HA.

The pressure inside the nozzle chamber was found to be a critical parameter, since it was the equilibrium between the nozzle chamber pressure and the HA solution injection rate that allowed the formation of the spray of particles. Stable spraying was possible for nozzle chamber pressures between 0.5 and 0.3 barg. Particles produced with lower pressures tended to be bigger, as shown in Figure 3(d), but the probability of nozzle clogging also increased, reducing the particle batch yield. Further than producing bigger particles, the decreasing of nozzle pressure seems to increase the size dispersion of the particles, resulting in a broader distribution of particle sizes for the batches produced with lower pressure. The distance between the nozzle and the dehydration/crosslinking bath also influenced the particle size, but the differences were only significant for distances too large; for those, the area in the base of the beam cone of sprayed particles was bigger than the area of the dehydration/crosslinking bath container, resulting in the loss of particles.

In summary, with the various parameters tested, the spray/dehydration process allowed the production of batches of particles with median sizes ranging from 7.17 µm, for the lower HA molecular weight, up to 55.74 µm for the higher concentration (2%) tested for the 830 kDa HA (Table 2).

HA has been proposed for the production of microparticles for sustained drug administration. In particular, HA microparticles with a mean size of 20–30 µm, prepared by solvent evaporation of HA esters⁴⁴ or by spray-drying,⁴⁵ were shown to have high mucoadhesive properties, and adequate drug loading ability for the delivery of intranasally administered drugs. The data herein presented show the possibility of producing such HA microparticles using a safer methodology, thus increasing its potential for several therapeutic applications.

HA microparticles degree of crosslinking

The methylene blue (MB) sorption assay was used to determine the concentration of carboxylic acid groups in HA microparticles batches. The anionic form of the HA carboxylic acid is negatively charged and the methylene blue cations can bind to the carboxylic anions. As methylene blue solution is added to the solution containing the sample, the MB binds to the carboxylate ion sites until the sites are saturated (Figure 4). The adsorption increases up to the saturation point, beyond which further addition of methylene blue does not result in additional methylene blue sorption, as seen in the slope of the amount of MB adsorbed. The percentage of non-covalently crosslinked anionic carboxylic moieties of the HA microparticle batches in the total number of carboxylic acid available, allowed the estimation of the degree of crosslinking (D_CL) of the HA batches produced with different concentrations of HA and different molecular weights.

As observed in Figure 4, the increase of HA concentration (Figure 4(a) to (d)) results in lower D_CL. Despite the absence of pyridine buffer, the crosslinking occurred, since the HA/ADH solution was at pH 4.0. The reaction between ADH and HA is strongly dependent on the pH and the amount of carbodiimide available.¹⁶ Thus, a molar excess of carbodiimide was used in all the reactions. Nevertheless, the CL reaction promoted after the diffusion of carbodiimide from the dehydration/crosslinking bath was hampered by the fast dehydration of the HA droplets. Since the diffusion rate depends on the solute concentration, the batches produced with increasing HA concentrations yielded proportionally lower D_CL. Comparing Figure 4(c) and (e), is possible to see the effect of the buffering of the crosslinking bath with pyridine. The buffering of the CL bath increased substantially the D_CL from 68% to nearly 99%, for batches produced in comparable conditions. Finally, the number of crosslinking steps was also analyzed. As previously observed in the FTIR spectra analysis (Figure 2(b)), batches submitted to 2 CL cycles present a more extended reaction, yielding a D_CL of 100% (Figure 4(f)).

Enzymatic degradability of the HA microparticles

The degradability of the different HA microsphere batches, produced with different HA molecular weights representative of small (66 kDa), medium (830 kDa), and high (2 MDa) molecular weight, was assessed by incubation of the microparticles (4 g per formulation) in 1 mL of a 1000 U/mL HAse solution or in PBS, for a period of 14 days (Figure 5). No degradation was observed during the 14 days of the experiment when the particles were incubated in PBS. On the other hand, as expected, the particles were degraded by incubation in 1000 U/mL HAse solution. The higher degradation was obtained for the microparticles produced with the 830 kDa HA, with a maximum of around 13% of degradation after 14 days, followed by the 2 MDa HA microparticles with around 1% of degradation, and the 66 kDa HA microparticles, for which almost no NAG release was detected. The higher degradation reported for the 830 kDa batch should be related with the lower D_CL calculated for this batch (68% while both the 2 MDa and 66 KDa HA had a D_CL of 100%). These observations confirm that the carboxylic groups in the β-glucoronic acid unit are the activation center of this enzyme and that total blockage of these groups can restrict the cleavage of beta (1–>4) glycoside bonds by this enzyme.^46–48

The HAase activity in the human body varies from tissue to tissue,⁴⁹ ranging from 0.0028 ± 0.0004 U/L in human plasma of healthy patients⁵⁰ to 38.5 U/mL in human ovaries.⁵¹ However, the HAase activity can be enhanced as response to injury or disease. In cases of patients suffering from coronary artery disease, for example, the HAase activity in human plasma significantly increases, reaching 3.8 ± 0.7 U/L.⁵⁰ In case of injury or disease, the degradation rate can be increased not only by the enhancement of HAses expression but also by the presence of reactive oxygen species.³ Moreover, the pH variation of injured tissues can influence the HA degradation mediated by HAses.^49,52 In this study, the concentration of HAase used for the degradation assay largely exceeded the normal physiologic conditions (about 5 × 10⁵ times the HAase activity in human plasma). It should be noted that these results only give information regarding relative degradation times and do not represent actual times for in vivo degradation. Given the in vivo expected degradability of HA matrix of the microparticles, namely by enzymes active in the remodeling of wounded tissues, and the potential of HA microparticles to release growth factors in a requested way,²⁰ the HA microparticles would be potential porogenic elements for the development of degradable CPC composites aiming the regeneration of bone defects. The low degradability inherent of the CPC is a drawback for their effectiveness in the regeneration of fully functional new bone⁵³ and this has been addressed through the incorporation of degradable microparticles of gelatin,⁵⁴ chitosan⁵⁵ or poly-l-glycolic acid (PLGA)⁵³ as sacrificial materials to produce highly interconnected porous structure.

HA microparticles swelling and PL loading ability

The graphic in Figure 6(a) represents the weight variation of HA microparticles incubated either with ddH₂O or PL for up to 21 h. It is evident an initial swelling of around 800 wt% for both conditions under study. Nevertheless, this value is only reached by microparticles immersed in PL after 15 min of incubation, followed by a significant reduction during the following 30 min and a slow stabilization of weight that occurs during the remaining 20 h of the assay. After stabilization, the swelling in ddH₂O reached 840%, while in PL it reached 710%. This phenomenon will be further discussed and might be explained by a swelling mechanism that occurs in sequential stages and includes different processes.

The in vitro release of PL proteins from HA microparticles was evaluated after loading HA microparticles with PL for 15, 30, 45, and 60 min, upon which the PL supernatant was removed. The loaded microparticles were incubated in PBS and the elution of proteins followed up to 24 h (Figure 6(b)). For all the loading times tested, the release profile was characterized by an initial burst of protein release followed by a sustained delivery of proteins, after 3 h of incubation. Regarding the total amount of protein released, it was found to be inversely proportional to the incubation time of the microparticles in PL. These results corroborate the trend observed in the swelling assay: a strong uptake during the first 15 min of the PL protein solution, followed by a significant weight reduction and stabilization over time.

The Korsmeyer–Peppas model allows for the prediction of the drug release model in polymeric systems, by analysis of the release kinetics of the first 60% of total protein loaded.⁴² The fitting of the release profile obtained in this study was biased by the difficulty to estimate the total protein loaded into the HA microparticles. The protein loading was not dependent on the initial protein concentration in PL, but on the incubation time. Thus, the total protein released was roughly estimated as the maximum amount of protein release over the 24 h of incubation. However PL is a complex cocktail of different proteins,^34,56 and despite the apparent exhaustion of solute, this approach disregards the amount PL proteins that could be more intensely interacting with the microparticle HA matrix. Therefore, the linear phase of the initial burst release, representing the first two hours of incubation, was analyzed. The value of the release exponent in PL protein release from HA microparticles ranged from 0.2 to 0.15 for the 15 min and 1 h of uptake time, respectively. This range of values is beyond the limits of Fickian release from a sphere (n = 0.432) predicted for the Korsmeyer–Peppas model.^57,58

In the case of polydisperse microparticles suspension, as the HA microparticles batches produced by spray/dehydration, n values for Fickian diffusion become 0.30.⁵⁹ An acceleration of drug transport at early times is attributed to the portion of particles smaller than the mean size and retardation at longer times to the portion of particles larger than the mean size.⁵⁹ Still, the simple diffusion of the PL proteins cannot explain the fast initial burst of protein release, according to the power law of Korsmeyer–Peppas. The erosional models could not also be applied, since the microparticles are only degraded under the action of HAases in the time frame of the release experiment. Since the particles were immediately ressuspended in the release media (ddH₂O) after the incubation in PL, the initial “burst” of release could be merely the interstitial protein remaining in the space between the microparticles, which was washed in the first incubation time. In fact, by fitting the protein release profile to the Kim and Fassihi⁴³ modified power-law model, for the time frame between the initial time point and the stationary- (lag-) phase, and discounting the protein burst before the first 15 min of incubation, it was possible to see that the protein release respected a Fickian release profile, tending n to ∼0.5 for all the loading periods tested.

Figure 6(d) schematizes the possible mechanism underlying this phenomenon. The dry HA microparticles have an internal trabecular structure that is rapidly filled with solutions in the first minutes after immersion, as already noticed in the swelling assay (Figure 6(a)). In this process, all the content of the solutions enters to the porous core of the microparticles. Meanwhile, the HA matrix of the microparticles trabecula starts to swell, reducing the size of the pores inside of the microparticles. The steric properties of the HA matrices⁶ creates a barrier for the entry of large molecules, and only the water and small molecules present in PL would contribute for the swelling of the HA meshes. The PL proteins will then adsorb to the surface of the trabecula by electrostatic interactions and become trapped inside of the closing pores of the microparticles. Since the PL proteins have different isoeletric points (pI), the electrostatic interactions and probability of remaining adsorbed to the HA microparticles, which are negatively charged at physiologic pH, will vary. The albumin, one of the main constituents of PL representing nearly 60% of the total protein dry weight,⁶⁰ with an acidic pI (at pH 4.7), and other negatively charged solutes will be gradually expelled from the microparticles incubated in PL solution until an equilibrium is reached, either by diffusion or by the reduction of available volume space after the swelling of HA microparticles trabeculae. Since the swell of the HA particles in ddH₂O is higher than in PL (Figure 6(a)), the trabeculae will become thicker and the protein solution trapped into the pores will be forced out of the HA particles in a higher rate than the simple diffusion (Figure 6(d)). On the other hand, most of the GFs present in PL with therapeutic interest have basic pI (TGF-β at pH 8.90; PDGF-A at pH 9.52; PDGF-B at pH 9.39; VEGF-1 at pH 8.66; FGF-2 at pH 9.6). These molecules are expected to bind electrostatically to the HA matrix and be further released in a sustained manner by ion exchange or by the degradation of the HA microparticles matrix promoted by HAses released for the ECM remodeling promoted during the wound healing process. Release of TGF-β and PDGF from GAS-produced HA microparticles was shown to be dependent upon the erosion of the particles, and not by diffusional processes.²⁰

In fact the PL growth factors TGF-β, PDGF and VEGF tend to bind preferentially to polyanionic polymers, reaching densities higher than in the PL mother solution.⁶¹ Oliveira and co-workers⁶¹ showed that it is possible to sequester the aforementioned factors into polyelectrolytes layer by layer, and the proportion of each factor is dependent on the sulfation degree of the polymer. Moreover, the coating of the polyelectrolyte surfaces with PL-derived factors modulated the adhesion and morphogenetic response of human adipose-derived cells.⁶¹ The use of HA microparticles for the focal sustained delivery of PL growth factors, with pro-angiogenic potential,⁶² would be of surplus importance, particularly in the treatment of ulcerated wounds in diabetic patients.²⁰

In summary, the results obtained suggested that the HA microparticles developed in this work can be used for the sustained delivery of cationic soluble factors with therapeutic interest in various clinical therapies or regenerative medicine applications.

Conclusions

In this work, we developed a new method to produce biodegradable hyaluronic acid microparticles, with a wide range of characteristics that can be accurately controlled/tailored to specific applications. More importantly, the proposed methodology presents several advantages over other previously described for the production of HA microparticles, namely: (1) it allows the production of large amounts of particles in a semi-continuous way; (2) it allows the fine-tuning of particles shape, morphology, and degradability; (3) it avoids the need of an oil phase and surfactants, thus the washing steps for the recovery of particles and removal of by-products are reduced, as well as the production of oil residues; and (4) it uses a crosslinking chemistry that occurs at mild temperature conditions and enables the use of native HA.

The microparticles produced using the spray/dehydration system developed in this work are stable in aqueous solutions, and are degraded only under extremely high concentrations of HAase in vitro. Moreover, the HA microparticles can be loaded with platelets-derived growth factors, and they release them in a controlled manner.

In sum, the HA microparticles developed in this study are expected to find valuable applications in regenerative medicine approaches, either providing biochemical cues for the endogenous cell migration and proliferation, or releasing in a controlled manner growth factors that support adequate tissue regeneration.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research leading to these results has received funding from Fundação para a Ciência e a Tecnologia (FCT) under project BIBS (PTDC/CVT/102972/2008), from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012-316331-POLARIS and from the project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” (RL1 – ABMR – NORTE-01-0124-FEDER-000016) cofinanced by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Pedro S. Babo acknowledges his FCT PhD grant SFRH/BD/73403/2010.

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