The Use of an Efficient Microfluidic Mixing System for Generating Stabilized Polymeric Nanoparticles for Controlled Drug Release

Abstract

Microfluidics is a promising system for efficiently optimizing the experimental conditions for preparing nanomedicines, such as self-assembled nanoparticles. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are promising drug carriers allowing sustained drug release. Here, we encapsulated the model drug curcumin, which has many pharmacological activities, into PLGA nanoparticles and investigated the effects of experimental conditions on the resulting PLGA nanoparticles using a microfluidics system with a staggered herringbone structure that can stir solutions through chaotic advection. The total flow rate and flow rate ratio of the solutions in the microfluidics system affected the diameters, polydispersity index, and encapsulation efficiency of the resulting PLGA nanoparticles and produced small, homogenous PLGA nanoparticles. The incorporation of polyethylene glycol (PEG)–PLGA into the PLGA nanoparticles reduced the particle size and improved the encapsulation efficiency. Initial burst release from the PLGA nanoparticles was prevented by the incorporation of PEG2000–PLGA. Curcumin-loaded PEGylated PLGA nanoparticles showed cytotoxicity similar to that of other formulations. This microfluidics system allows high throughput and is scalable for the efficient preparation of PLGA nanoparticles and PEGylated PLGA nanoparticles. Our results will be useful for developing novel PLGA-based polymer nanoparticles by using the microfluidics.

Nanomedicine is the application of nanotechnology to medicine and is one approach for the generation of novel functional drugs. Various nanomedicines have entered clinical trials globally¹⁾ and food and drug administrations have to date approved over 50 formulations such as liposomes, polymeric nanoparticles, drug nanoparticles, and metal-based nanoparticles.²⁾ These nanomedicines are mainly designed to improve the therapeutic effects and decrease the side effects of the therapeutic agent due to enhanced drug delivery.

Poly(lactic-co-glycolic acid) (PLGA) is used in various controlled and sustained drug release strategies. PLGA, a biocompatible and biodegradative polymer, is composed of lactic acid and glycolic acid and is hydrolyzed into acids that are believed to enter the Krebs cycle.³⁾ PLGA-based materials can be used to encapsulate both hydrophilic and lipophilic drugs. There are many kinds of PLGA-based materials, such as PLGA microparticles containing the luteinizing hormone-releasing hormone superagonist leuprorelin acetate,⁴⁾ PLGA implants for treating eye diseases,⁵⁾ PLGA bioresorbable surgical sutures,⁶⁾ and PLGA scaffolds for tissue engineering.⁷⁾ PLGA nanoparticles are a promising injectable drug form for intravenous administration. The surface modification of PLGA nanoparticles by polyethylene glycol (PEG) results in the formation of a water layer, allowing the nanoparticles to escape recognition by mononuclear phagocyte system components such as macrophages, resulting in a prolonged blood circulation time. A prolonged blood circulation residency time of PEGylated PLGA nanoparticles leads to passive accumulation in tumor tissue due to the leaky tumor vasculature, and this is called the enhanced permeability and retention effect.⁸⁾ The application of PLGA nanoparticles has been studied for the treatment of various diseases, such as bone infection,⁹⁾ arteriosclerosis,¹⁰⁾ neurological disorders,¹¹⁾ cancer,¹²⁾ and for use as vaccines.¹³⁾

Appropriate infrastructure for a manufacturing process is important for the efficient manufacture of nanomedicines. Laboratory-scale experiments involving trial and error are frequently conducted to create the desired nanoparticles. However, methodologies are required that efficiently both optimize the formulation and can be scaled up. Thus, the use of high throughput equipment can save time and increase handling efficiency. However, the preparation of nanomedicines is complex and requires gentle conditions. Furthermore, injectable nanomedicines are manufactured using processes different from those for typical injection formulations. Formulation parameters, such as physicochemical properties (diameter, poly dispersity index) and encapsulation efficiency are indexes of the quality of a nanomedicine. The special equipment typically used in laboratories is not always suitable for scale-up. Scale-up often results in substandard nanomedicine products, such as larger and heterogeneous nanoparticles which induce Ostwald ripening, making the formulations unstable, with poor batch-to-batch uniformity and reproducibility.

In this study, we focused on the preparation of a drug-loaded PLGA nanoparticle formulation using a NanoAssemblr system (Fig. 1). NanoAssemblr is a micro-channel-based system for creating nanoparticle-based drugs. Many kinds of micro-channel-based systems have been developed.^14–17) The staggered herringbone structure in the microchannels of the NanoAssemblr system stirs the solution with chaotic advection,^18,19) contributing to the preparation of homogeneous and reproducibly self-assembling nanoparticles. The generation of self-assembled nanoparticles using this device is promising. Examples include lipid nanoparticles,²⁰⁾ polymer nanoparticles,^21–23) non-ionic surfactant nanoparticles²⁴⁾ and cochleate microparticles.²⁵⁾ To date, most applications have focused on the preparation of liposomes^26–28) and there is little information regarding the generation of other types of nanoparticles using this approach. In this study, we focused on the preparation of PLGA nanoparticles and investigated the influence of various conditions on the PLGA nanoparticles.

Fig. 1. Scheme Showing the Preparation of PLGA Using the Microfluidic System Employed in the Current Study

MATERIALS AND METHODS

Materials

PLGA (Resomer^® series) and PEG–PLGA were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.), as shown in Table 1. Polyvinyl alcohol (PVA), polyoxyethylene sorbitan mono-oleate (Tween 80) and sodium cholate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Curcumin was purchased from Tokyo Kasei Industry Co. (Tokyo, Japan). All other chemicals were reagent grade and were used without purification.

Table 1. Information Regarding the PLGAs Used in the Present Study

(A) PLGA
Name	RG 752H	RG 502H	RG 504H
L/G ratio	75/25	50/50	50/50
M.W.	4000–15000	7000–17000	38000–54000
(B) PEG–PLGA
Name	PEG2000	PEG5000
L/G ratio	50/50	50/50
M.W. (PLGA)	11500	7000
M.W. (PEG)	2000	5000

(A) PLGA and (B) PEG–PLGA. Molecular weight (M.W.).

Preparation of PLGA Nanoparticles Using NanoAssemblr

PLGA nanoparticles were prepared using a NanoAssemblr Benchtop Instrument (Precision NanoSystems, Vancouver, BC, Canada) following the manufacturer’s instructions. Briefly, a syringe of acetone solution containing 1 mg/mL curcumin and 10 mg/mL PLGA (RG 752H, RG 502H, or RG 504H) and another syringe of aqueous solution containing 1% stabilizers (PVA, Tween 80, or sodium cholate, w/v) were attached to the instrument. PLGA (RG 502H) and PEG–PLGA (PEG2000 or PEG5000) were dissolved in acetone to provide PLGA (100%) and PEG–PLGA (0, 2.5, 5, 10 or 20% (w/w) against PLGA) solutions prior to preparing the PEGylated PLGA nanoparticles. The solutions were dispensed into the microfluidic cartridge, and mixed by stirring in the staggered herringbone structure, as shown in Fig. 1. Parameters such as the total flow rate and flow rate ratio were controlled by a computer linked to the system. The product mixture containing curcumin-loaded PLGA nanoparticles was collected in a plastic tube set in the instrument. Following 100-fold dilution of the sample solution with pure water, the mean diameter and polydispersity index (PDI) of the PLGA nanoparticles were measured using a Zetasizer Nano-S instrument (Malvern Instruments Ltd., Malvern, U.K.). The influence of organic solvent on the analysis by dynamic scattering method is minimum due to the dilution. The samples were ultracentrifuged (20000 rpm, 10 min, 4°C) to remove the supernatant and wash the samples by water. The samples were frozen at −80°C, freeze-dried, then stored until use.

Determination of the Curcumin Concentration

The amount of curcumin in the nanoparticles was determined by measuring the absorbance of the solution at 423 nm using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) as described previously.²⁹⁾ The encapsulation efficiency of curcumin in the PLGA nanoparticles was calculated from the concentration of curcumin in nanoparticles and in supernatant after ultracentrifuge as described in Preparation of PLGA Nanoparticles Using NanoAssemblr.

Scanning Electron Microscopy (SEM)

Solutions of curcumin-loaded PLGA (RG 502H) nanoparticles were filtered through a 0.2 µm membrane and then freeze-dried. The appearance of each sample powder was observed using a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan).

Differential Scanning Calorimetry

Approximately 2 mg of sample was placed in an aluminum pan and crimped shut. The samples were analyzed using a Shimadzu DSC-50 (Shimadzu) in the temperature range at 10°C/min.

In vitro Drug Release

The total flow rate was fixed at 6 mL/min and the flow rate ratio was fixed at 3/1 to prepare curcumin-loaded PLGA nanoparticles. Five percent of PEG–PLGA was incorporated to prepare PEGylated PLGA nanoparticles. Drug (curcumin) release from PLGA nanoparticles was measured as previously reported, with some modifications.³⁰⁾ Briefly, 1.6 mg of PLGA nanoparticles was dispersed in 8 mL of 0.3% Tween 80 aqueous solution, then 1 mL samples were transferred into microtubes. The samples were incubated at 37°C and samples were removed at appropriate time points (6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 14 d). Each sample was ultracentrifuged (20000 rpm, 10 min, 4°C) and the supernatant (900 µL) was collected. Supernatant (50 µL) was mixed with 950 µL of acetone and the absorbance was measured by UV-Vis as above (423 nm).

Cell Culture

The human lung adenocarcinoma epithelial cell line A549 was obtained from ATC C (Manassas, VA, U.S.A.). The cells were grown in medium composed of high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. The cells were maintained in 10 cm dishes filled with medium at 37°C and under a 5% CO₂ humid atmosphere.

Cytotoxicity Assay

RG 502H was used to prepare PLGA nanoparticles. The total flow rate was fixed at 6 mL/min and the flow rate ratio was fixed at 3/1 to prepare curcumin-loaded PLGA nanoparticles. Five percent of PEG–PLGA was incorporated to prepare PEGylated PLGA nanoparticles. The WST assay was used to evaluate cytotoxicity as previously reported.²⁹⁾ Briefly, cells were seeded into a 96-well plate at a density of 5000 cells/well. After preculture for 24 h, curcumin formulations were added to individual wells to a concentration of 50 µM. The same amount of formulation without curcumin was added to wells as control. After exposure to the formulation for 24 h, the cells were washed with phosphate buffered saline, and medium containing a 10% solution of Cell Counting Kit solution (Dojindo Laboratories, Inc., Kumamoto, Japan) was added to each well. After incubation for 1 h, the absorbance of each well was measured using a Wallac 4000 ARVO multi-label counter (wavelength, 450 nm; PerkinElmer, Inc., Waltham, MA, U.S.A.). Cytotoxicity was determined by calculating the reduced viability of control (untreated) cells.

RESULTS AND DISCUSSION

Characteristics of Curcumin-Loaded PLGA Nanoparticles

The characteristics of PLGA nanoparticles prepared using the present microfluidics system are shown in Fig. 2. We used curcumin as a model drug and it was loaded into PLGA nanoparticles. Curcumin has many pharmacological effects and has been studied for the treatment of many diseases, such as inflammatory bowel diseases,³¹⁾ renal fibrosis,³²⁾ wound healing,³³⁾ and cancer.^34,35) However, the poor bioavailability and stability in serum pumper the therapeutics effect. The nanoparticulation of curcumin is one of the efficient strategies to improve the drug dissolution and bioavailability.

Fig. 2. Characteristics of PLGA Nanoparticles Prepared Using the Microfluidics System

(A) Particle distribution, (B) SEM image, and (C) DSC thermograms. RG 502H was used to prepare representative PLGA nanoparticles. 1% PVA was used as the stabilizer. The total flow rate was fixed at 6 mL/min and the flow rate ratio was fixed at 3/1.

The particle size distribution data of curcumin-loaded PLGA nanoparticles showed a single peak and the average size was around 200 nm (Fig. 2A), suggesting that the resulting PLGA nanoparticles are monodispersed fine particles. Image of the resulting PLGA nanoparticles on the experimental conditions is shown in Fig. 2B. Homogenous spherical PLGA nanoparticles were formed. The differential scanning calorimetry (DSC) scans of PLGA, PLGA nanoparticles, and curcumin-loaded PLGA are shown in Fig. 2C. The incorporation of curcumin into PLGA nanoparticles affected the shift of peak temperature which meant the glass-transition temperature.

The Influence of Formulation Parameters on Curcumin-Loaded PLGA Nanoparticles Produced by the Microfluidic Device

Next, we investigated the influence of experimental conditions and the composition of the PLGA nanoparticles on the resulting curcumin-loaded PLGA nanoparticles in order to comprehensively understand the capabilities of the microfluidic device for this application. As shown in Fig. 3, the effects of stabilizers on mean diameter, PDI, and encapsulation efficiency were investigated using three stabilizers commonly used for the preparation of PLGA nanoparticles. PLGA nanoparticles that were monodisperse and approximately 200 nm in diameter were prepared by using PVA. In contrast, the particle size of PLGA nanoparticles prepared with sodium cholate was much smaller and the encapsulation efficiency showed high deviation. PLGA nanoparticles prepared with Tween 80 were smaller and the encapsulation efficiency was much lower than nanoparticles prepared using PVA. The information of stabilizer was shown in Table 2. While hydrophilic/lipophilic balance is almost the same among the stabilizers, the critical micelle concentration value is different (PVA (no value)>sodium cholate>Tween 80). So, we think that other factors in addition to these can affect the current results. Since PVA provided the highest encapsulation efficiency, it was used as the stabilizer in subsequent experiments.

Fig. 3. Effect of Stabilizer on the Resulting Curcumin-Loaded PLGA Nanoparticles

(A) Particle size, (B) PDI, and (C) encapsulation efficiency. The total flow rate was fixed at 6 mL/min and the flow rate ratio was fixed at 3/1. The data represent the mean±standard deviation (S.D.) (n=3).

Table 2. Information Regarding the Stabilizers

	Hydrophilic/lipophilic balance (HLB)	Critical micelle concentration (CMC)
PVA	18	No data
Sodium cholate	18	0.70%†
Tween 80	15	0.0022%††

^†: The data was calculated from the literature (16.2 mM≒0.70%).^{44) ††}: The data was collected from the literature.⁴⁵⁾

We then tested three PLGAs with different L/G ratios and molecular weights, as shown in Table 1A. The use of different PLGAs is expected to affect drug release, as described below. The influence of the total flow rate of the PLGA/curcumin acetone solution and the PVA aqueous solution was investigated (Fig. 4). A similar effect on particle size, PDI, and encapsulation efficiency was found for the different PLGAs. The mean diameters of the curcumin-loaded PLGA nanoparticles decreased as the total flow rate increased, with the diameters being less than 200 nm at higher flow rate. In contrast, the PDI of the PLGA nanoparticles increased as the total flow rate increased. Homogeneous PLGA-nanoparticles were obtained at the slowest total flow rate under these experimental conditions. The encapsulation efficiency of curcumin decreased as the total flow rate increased and was highest at the slowest total flow rate.

Fig. 4. Effect of Total Flow Rate on the Resulting Curcumin-Loaded PLGA Nanoparticles

(A) Particle size, (B) PDI, and (C) encapsulation efficiency. The flow rate ratio was fixed at 3/1. The data represent the mean±S.D. (n=3).

Regarding the production of PLGA nanoparticle, PLGA nanoparticles are prepared by the emulsion solvent dispersion (also called solvent displacement method).³⁶⁾ After the mixing PLGA/curcumin organic solution with stabilizer aqueous solution in microfluidics, organic nanoemulsion is produced. PLGA starts to crystalize on the water/solvent interface as well as curcumin, and accordingly, curcumin is incorporated into PLGA nanoparticle as drug–PLGA matrix. Curcumin which has very low aqueous solubility is prone to be crystalized before PLGA do. There is a tendency that smaller nanoparticles have lower encapsulation efficiency due to the smaller space in the particles as it is observed in Figs. 4A and C.

Several earlier studies demonstrated the preparation of liposomes using a NanoAssemblr. Guimarães Sà Correia et al. reported that the diameters of liposomes decreased when the total flow rate was changed from 1 to 6 mL/min and little change in diameter was obtained when the total flow rate was changed from 6 to 20 mL/min.³⁷⁾ Kastner et al. showed a slight decrease in the diameters of liposomes by increasing the total flow rate from 0.5 to 2 mL/min under several experimental conditions.²⁷⁾ The same group obtained liposomes with similar diameters when the total flow rate was changed from 2 to 6 mL/min.²⁸⁾ Consequently, it appears that the diameters of liposomes can be changed by using a total flow rate of less than 2 mL/min. In our case, the diameter of the PLGA nanoparticles changed slightly between 3 to 12 mL/min (Fig. 4A). These results suggest that the flow rate required to change the diameter of the nanoparticles is different for each type of self-assembled nanoparticle. Hybrid nanoparticles with a PLGA core were previously prepared using a microfluidics system²¹⁾ and showed a remarkable decrease in diameter with increasing total flow rate from 5 to 20 mL/min. These results partially support our present results. An increase in total flow rate increased the heterogeneity of the particles (Fig. 4B). Joshi et al. reported that the PDI of liposomes increased when the total flow rate was increased from 10 to 15 mL/min.²⁶⁾ This increased PDI resulting from an excessively high total flow rate might inhibit the formation of PLGA nanoparticles, resulting in decreased particle diameter and lower drug encapsulation efficiency (Fig. 4C).

Next, the flow rate ratio of the PLGA/curcumin acetone solution into the PVA solution (organic solution/aqueous solution) was changed to understand the effect of the proportion of the organic solvent on mixing during PLGA nanoparticle formation (Fig. 5). The resulting curcumin-loaded PLGA particles had an average diameter of 200 nm, except for particles prepared using RG 504H at an increased amount of aqueous solution (5/1). The PLGA nanoparticles showed slightly increased PDI values as the proportion of aqueous solution increased, and the order of PDI was RG 752H=RG 502H<RG 504H. The encapsulation efficiencies of RG 752H and RG 502H were similar or decreased as the proportion of aqueous solution increased. In contrast, the encapsulation efficiency of RG 504H increased unexpectedly. Although we do not have clear reasons, the large molecular weight of RG504 may be crystalized fast after the mixing. The fast process of nanoparticulation of RG504 can help and control the encapsulation of curcumin, while the process led the smaller diameter by fast change of solvent component.

Fig. 5. Effect of Total Flow Rate Ratio on the Resulting Curcumin-Loaded PLGA Nanoparticles

(A) Particle size, (B) PDI, and (C) encapsulation efficiency. The total flow rate was fixed at 6 mL/min. The data represent the mean±S.D. (n=3).

PLGA nanoparticles are prepared, following the mechanism of emulsion solvent diffusion method as described above. The different flow rate ratio can affect the rate of diffusion between water and miscible organic solvent, water solubility of PLGA and drug, and the rate of crystallization. The decrease of proportion of organic solvent increased the particle size of PLGA nanoparticles in other report,³⁸⁾ which is evidence that PLGA is prepared by the process of crystallization. In contrast, there is different tendency about the flow rate ratio when liposome was prepared as described below. This means that liposome has different mechanism about its formation. In this study, the drastic change of flow rate ratio did not lead the change of particle size compared with typical stirring (such as³⁸⁾). Additionally, PLGA nanoparticles generated using other microfluidics systems showed increased diameters upon changing the flow rate ratio from,³⁹⁾ providing other example of different tendencies using different microfluidics systems. So, this microfluidic device probably has a strong advantage to control the size.

Several groups have shown that the flow rate ratio of organic solution to aqueous solution is a key factor in the preparation of liposome formations. For example, changing the flow rate ratio to above 1/1 (2/1,²⁸⁾ 3/1^26,27)) resulted in a remarkable decrease in the diameters of liposomes with several compositions. These results suggest that dilution of the organic solution into the aqueous solution is remarkably promoted by increasing the flow rate ratio. In contrast, when the flow rate ratio was 1/1, it could take time to reduce the solubility of the lipids after mixing the solutions and the particles were formed slowly, thereby producing liposomes of larger size. The diameters of liposomes obtained using different microfluidics devices were also decreased by changing the flow rate ratio.⁴⁰⁾ In accordance with the above discussion regarding the relationship between PDI and flow rate ratio, an increase in PDI was observed by increasing the flow rate ratio (Fig. 5B), consistent with several previous articles^27,28) but the opposite of that reported in another study.²⁶⁾ We believe that an excessively high flow rate ratio, which dramatically reduces the proportion of organic solution and affects the solubility of the carrier (PLGA in our case and phospholipids in the other reports), may produce heterogeneous nanoparticles. Several researchers have investigated other parameters, such as the concentration of the carriers, buffers, and solvents, which might influence solubility and stability of the carrier and the characteristics of the nanoparticles.^24,26) Additionally, the viscosity is a possible key parameter. Although we did not investigate it, the concentration of PLGA and drug may affect the viscosity and affect the particle size and PDI.

Characteristics of PEGylated Curcumin-Loaded PLGA Nanoparticles Produced by the Microfluidic Device

PEGylated curcumin-loaded PLGA nanoparticles were prepared by mixing PLGA and PEG–PLGA/curcumin solution. Two types of PEG–PLGAs with different molecular weight PEGs were used for the preparation of PEGylated liposomes, as shown in Table 1B. As shown in Fig. 6A, we found that the mean diameters of PEG2000-incorporated PLGA nanoparticles slightly decreased as the amount of PEG–PLGA incorporated increased, while PEG5000-incorporated PLGA nanoparticles showed little change despite the use of increased amounts of PEG–PLGA. PDI tended to be slightly smaller when a larger amount of PEG2000 was incorporated into the PLGA (Fig. 6B). The incorporation of an increased amount of PEG2000 into PLGA nanoparticles resulted in decreased encapsulation in both the PEG5000 and PEG2000–PLGA nanoparticles (Fig. 6C). Interestingly, the encapsulation efficiency of curcumin into the PEGylated PLGA nanoparticles was remarkably higher than that into non-PEGylated PLGA nanoparticles (0% incorporation of PEG–PLGA).

Fig. 6. Effect of the Incorporated PEG–PLGA on the Resulting Curcumin-Loaded PLGA Nanoparticles

(A) Particle size, (B) PDI, and (C) encapsulation efficiency. The total flow rate was fixed at 6 mL/min and the flow rate ratio was fixed at 3/1. The data represent the mean±S.D. (n=3).

PEGylated PLGA has surface active properties similar to that of surfactant and thus acts as a stabilizer, as described above (Fig. 3). We estimated that the increased amount of incorporated PEG–PLGA provides conditions both for self-assembly and the entrapment of drug, resulting in optimal PLGA nanoparticles. We suppose that the addition of other stabilizers (PEG–PLGA in this case) can collaborate the improvement of encapsulation efficiency of PLGA nanoparticles supplementary. For example, the incorporation of d-α-tocopheryl polyethylene glycol 1000 succinate into PLGA nanoparticle could improve the encapsulation efficiency of paclitaxel.⁴¹⁾ This reference can partially support our results (Fig. 6C). PLGA with short PEG chains generated smaller diameter PLGA nanoparticles (Fig. 6A), although increased amount of PEGylation into PLGA nanoparticle led low drug encapsulation efficiency (Fig. 6C). A slight increase in the water-solubility of PEG–PLGA polymer appears to aid the preparation of nanoparticles in our study. In contrast, the particle size of folate-conjugated PEGylated liposomes showed different tendency when different amounts of PEG-lipid were incorporated into liposomes using different microfluidics systems.⁴²⁾

The release of curcumin from PLGA nanoparticles was evaluated (Fig. 7A). The order of increasing release rate was RG 752H>RG 502H>RG 504H. An initial burst release was observed for all preparations and the amount of released drug was different for all PLGAs. The initial burst release was efficiently inhibited by incorporating PEG2000–PLGA (Fig. 7B) and the inhibitory effect of PEG2000–PLGA was larger than that of PEG5000–PLGA.

Fig. 7. Drug Release Profiles of Various (A) Curcumin-Loaded PLGA Nanoparticles and (B) Curcumin-Loaded PEGylated PLGA Nanoparticles

The data represent the mean±S.D. (n=3).

The degradation properties of PLGA are dependent on the L/G ratio and molecular weight. PLGAs with an L/G ratio of 1/1 result in a faster drug release profile. For example, Hung et al. reported that PLGA nanoparticles (50 : 50, L/G) showed a faster fluorescein release profile compared with nanoparticles (85 : 15, L/G) formed using different microfluidics systems.³⁹⁾ In contrast, the inverse pattern was observed in the present study (Fig. 7A) for reasons currently unclear. One possible reason is that the redispersibility of PLGA nanoparticle (i.e., the diameter after the dispersion of freeze-dried sample) may affect the drug release profile. On the other hand, other researchers have reported that the same liposome formulation subjected to different experimental conditions using a NanoAssemblr exhibited remarkably different drug release profiles.³⁷⁾ Focusing on the drug release profile of PEGylated PLGA, Khalil et al. prepared PEGylated PLGA nanoparticles using different methods and showed that PEGylation promoted drug release.³⁰⁾ Given their report, we believe that the preparation process, encapsulation efficiency, and other factors may affect drug release from PLGA nanoparticles. Using our experimental conditions, PEG2000-incorporated PLGA nanoparticles showed the slowest drug release (Fig. 7B). Given the sustained drug release from PEG2000-incorporated PLGA nanoparticles, we believe that the efficient encapsulation of curcumin into PEGylated PLGA particles will result in sustained drug release.

Next, we investigated the cytotoxic effect of curcumin-loaded PLGA nanoparticles using the A549 lung cancer cell line (Fig. 8). Curcumin powder showed a moderate cytotoxic effect. Since curcumin is a poorly water-soluble compound, it did not dissolve in the medium efficiently. Curcumin was dissolved with dimethyl sulfoxide (DMSO) to provide an alternative formulation. This curcumin solution showed a remarkable cytotoxic effect. However, a curcumin organic solution is unstable in medium, resulting in the crystallization of curcumin during 24 h incubation, thereby limiting the formulation of this poorly water-soluble drug. PLGA nanoparticles without curcumin (PLGA placebo) showed a slight cytotoxic effect. Our previous study reported that PLGA nanoparticles could suppress osteoblast cell proliferation and the secretion of bone formation marker in vitro because acidification of the medium can have a negative effect on cells.⁴³⁾ The curcumin-loaded PLGA nanoparticles exhibited a cytotoxic effect similar to that of curcumin solution. The released curcumin outside and/or inside the cells before and after endocytosis could affect cytotoxicity. Both curcumin-loaded PEG2000-incorporated PLGA nanoparticles and PEG5000-incorporated PLGA nanoparticles showed cytotoxic effects similar to non-PEGylated PLGA nanoparticles containing curcumin and curcumin solution. The results indicate that sustained drug release during 24 h incubation could provide similar cytotoxic effects. The cytotoxic effects of PEGylated PLGA nanoparticles are a useful property in vitro, and such formulations may be useful for increasing the residency time of nanoparticles in the blood circulation and their passive accumulation in tumor tissue in vivo, although further investigation is necessary.

Fig. 8. Cytotoxic Effects of Curcumin Formulations and PLGA-Based Formulations

The data represent the mean±S.D. (n=3).

CONCLUSION

Here we described the preparation and characterization of PLGA nanoparticles and PEGylated PLGA nanoparticles. Both the composition of the PLGA nanoparticles and the microfluidics system parameters remarkably affected the characteristics of the particles, and homogenous PLGA nanoparticles were obtained using this microfluidics system. The exploration, identification and optimization of important experimental conditions can be efficiently conducted using a small scale, high-throughput system. Although the original characteristics of the microfluidics system still require clarification, the current results will be useful for preparing PLGA nanoparticles using microfluidics systems.

Acknowledgments

We thank Mr. Kinya Kurihara (Nepa Gene Co., Ltd.) for assistance and technical advice regarding use of the NanoAssemblr. We also thank Mr. Hiroshi Takase (Graduate School of Medical Science and Medical School, Nagoya City University) for assistance and technical advice regarding use of SEM.

Conflict of Interest

The authors declare no conflict of interest.

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