Supercritical Fluid-Assisted Decoration of Nanoparticles on Porous

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Supercritical Fluid-Assisted Decoration of Nanoparticles on Porous Microcontainers for Co-Delivery of Therapeutics and Inhalation Therapy of Diabetes Ranjith Kumar Kankala, Xiao-Fen Lin, Hufan Song, ShiBin Wang, Da-Yun Yang, Yu Shrike Zhang, and Ai-Zheng Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00992 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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ACS Biomaterials Science & Engineering

Supercritical Fluid-Assisted Decoration of Nanoparticles on

Porous

Microcontainers

for

Co-Delivery

of

Therapeutics and Inhalation Therapy of Diabetes Ranjith Kumar Kankala,†, ‡,⊥Xiao-Fen Lin,†, ‡,⊥Hu-Fan Song,† Shi-Bin Wang, †, ‡ Da-Yun Yang,§ Yu Shrike Zhang,*,± and Ai-Zheng Chen*,†, ‡

†

Institute of Biomaterials and Tissue Engineering, Huaqiao University, Xiamen 361021, P. R.

China ‡

Fujian Provincial Key Laboratory of Biochemical Technology (Huaqiao University), Xiamen

361021, P. R. China §Fujian

Key Laboratory for Translational Research in Cancer and Neurodegenerative

Diseases, Institute for Translational Medicine, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, China ±

Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital,

Harvard Medical School, Cambridge, MA 02139, USA

* Corresponding Authors: Emails: [email protected] Tel./fax: +86 592 616 2326 (A.-Z.C.), [email protected] Tel.: +1 617 768 8221 (Y.S.Z). ⊥ R.K.K.

and X.F.L. contributed equally to this work.

1 Environment ACS Paragon Plus

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

The impact of nanotechnology and its advancements have allowed us to explore new therapeutic modalities. To this end, we designed nanoparticles-laden porous microparticles (NIPMs) co-loaded with small-interfering ribonucleic acid (siRNA) and glucagon-like peptide-1 (GLP-1) using the supercritical carbon dioxide (SC-CO2) technology as an inhalation delivery system for diabetes therapy. siRNA-encapsulating chitosan (CS) nanoparticles were first synthesized by an ionic gelation method, which resulted in particles with small size range (100150 nm), high encapsulation efficiency (~94.8%), and sustained release performance (~60% in 32 hours). These CS nanoparticles were then loaded with GLP-1-dispersed poly-L-lactide (PLLA) porous microparticles (PMs) by SC-CO2-assisted precipitation with the compressed antisolvent (PCA) process. The hypoglycemic efficacy of NIPMs administered via pulmonary route in mice persisted longer due to sustained release of siRNA from CS nanoparticles and the synergistic effects of GLP-1 in PMs, which significantly inhibited the expression of dipeptidyl peptidase-4 messenger ribonucleic acid (DPP-4-mRNA). This eco-friendly technology provides a convenient way to fabricate nanoparticle-microparticle composites for co-delivery of a gene and a therapeutic peptide, which will potentially find widespread applications in the field of pharmaceutics.

KEYWORDS: supercritical carbon dioxide technology, pulmonary delivery, small-interfering ribonucleic acid (siRNA), glucagon-like peptide-1 (GLP-1), diabetes mellitus


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INTRODUCTION Diabetes mellitus has become a significant health concern in the past few decades globally as approximately one in every five individuals is diagnosed with it.1-3 This metabolic disorder occurs due to increased blood glucose level caused by a pancreatic failure in releasing insulin from the β-cells (Type 1) or by insulin resistance from the body tissues (Type 2),4 which result in severe complications such as diabetic ketoacidosis, cardiac stroke, and chronic kidney disease, among others.5-7 Currently, the preferred treatment modalities of diabetes include direct delivery of insulin or other synthetic insulin sensitizers, which can enhance the release of insulin from the pancreas.4 Despite the success in the control of glucose levels in the blood, self-administration of insulin often leads to hypoglycemia, leading to severe consequences such as unconsciousness, coma, and eventually brain damage.8 On the other hand, the synthetic sensitizers of insulin also result in adverse effects, including hematologic abnormalities, liver toxicity, and hypoglycemia; however, no life-threatening effects have been reported so far.2,

9-10

To overcome these

limitations, an efficient delivery platform based on the utilization of natural sensitizers can be explored to efficiently control hyperglycemia. Glucagon-like peptide (GLP)-1 and its analogs, such as Exendin-4,11 are amongst hypoglycemic agents, not only sensitizing the release of insulin in blood12 but also exhibiting the extra-pancreatic effects that are effective in hypoglycemia such as reduced obesity-related cardiovascular risk, minimized appetite, restrained secretion of glucagon after a meal, reduced output of glycogen, slowed down gastric emptying, and improved β-cell function.13-16 However, the utilization of GLP-1 has been limited as it is quickly degraded by the dipeptidyl peptidase-4 (DPP-4) enzyme in plasma resulting in its short half-life in vivo for only 2-6 min.2, 17 This limitation can be predominantly surpassed by inhibiting the synthesis of a DPP-4 enzyme by downregulating the expression of its preceding mRNA through the small-


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interfering ribonucleic acid (siRNA)-based gene silencing effect.18 More often, the RNA interference-based gene knockdown technology is utilized to monitor the consequences of genetic transcription and can restrain the expression of corresponding genes.19-21 With numerous advantages such as high specificity, effectiveness, and endurance, RNA interference is suitable to effectively suppress the DPP-4-mRNA expression.22 The lung has become an attractive site for drug delivery as it is suitable for the absorption of many drugs due to numerous advantages such as non-invasive administration via inhalation aerosols, availability of high surface area for local and systemic absorption of drugs, receipt of the entire cardiac output, as well as lack of the first-pass metabolism, among others.7, 23-26 In this framework, porous microparticles (PMs) are one of the effective carriers for delivering sensitive biomolecules such as proteins or genes via pulmonary route of administration, because they are light enough to aerosolize and penetrate deeply into the lungs, and their large sizes prevent phagocytosis by lung macrophages.26-30

Comparatively, porous structures can rapidly

disintegrate in lungs and deliver therapeutic moieties better than solid microparticles.31-32 Despite the advantages and delivery efficacy, these PMs suffer from a few limitations such as poor encapsulation efficiency of drugs and rapid drug release.33 Spraying drying, emulsion-solvent evaporation, milling, and supercritical fluid (SCF) are the processing methods that are available to fabricate inhalation PMs.26, 29, 34-36 Amongst them, the SCF technology has gained enormous attention due to numerous advantages such as precise and convenient control over the particle morphology by tuning the operating conditions, low levels of remnant organic solvents in the end products, uniform particle size distribution, and protection of sensitive biomolecules.30, 37-38 In addition, the selection of excipients is relatively wide compared to other traditional techniques, as SCFs are highly compatible with most of the pharmaceutical



additives utilized for inhalation formulations.26,

29

Thus, this technology is potentially more

promising in preparing pharmaceutical formulations that are based on therapeutic peptides for inhalation drug delivery.26, 29

Figure 1. Schematic diagram showing SCF-assisted decoration of nanoparticles on porous microparticles for co-delivery of diabetic therapeutics.

Inspired by these facts, this study demonstrates the utilization of the SC-CO2 technology, in particular the precipitation with compressed anti-solvent (PCA) process, for the fabrication of nanoparticles-inlaid PMs (NIPMs) for synergistic therapy of diabetes. As shown in Figure 1, initially, siRNA-encapsulated chitosan (CS) nanoparticles were fabricated through an ionic gelatin method, and they were subsequently subjected to supercritical treatment, where the


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nanoparticles were loaded with the contemporaneously formed GLP-1-immobilized PMs. We then investigated various physicochemical attributes of the NIPMs such as morphology, drug loading efficiency, their release, and biosafety as well as their hypoglycemic effects in diabetic mice. The resultant hypoglycemic effect of NIPMs was not only due to the released GLP-1 from PMs but also contributed by the downregulation of DPP-4-mRNA by siRNA delivery from the CS nanoparticles, through the synergistic effects of the co-delivery system.

MATERIALS AND METHODS Materials GLP-1 was purchased from the China Peptides (Shanghai, China). Poly-L-lactide) (PLLA) (molecular weight: 50,000) was purchased from the Jinan Daigang Co. Ltd. (Jinan, China). Chitosan, pluronic F-127 (PF-127) and ammonium bicarbonate (AB) were purchased from Sigma-Aldrich (USA). HepG2 cells were obtained from cell bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM) was purchased from the Invitrogen Co. Ltd (Camarillo, USA). Fetal bovine serum (FBS) was purchased from the Hyclone Co. Ltd, (Logan, USA). siRNA (sense strand: 5’-GCUGCUCAAUGCUGAACAUTT3’; antisense strand: 5’-AUGUUCAGCAUUGAGCAGCTT-3’) specific to human DPP-4mRNA was obtained from Gene Pharma Co. Ltd (Shanghai, China). Dichloromethane (DCM, 99.8% purity) was purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). CO2 (99.9% of purity) was supplied by the Rihong Air Products Co. Ltd. (Xiamen, China). Fluorescein isothiocyanate (FITC) was purchased from the Sangon Biotech Co. Ltd. (Shanghai, China). The reagents of quantitative real-time polymerase chain reaction (QRT-PCR) were purchased from the Biyuntian Co. Ltd. and Takara Co. Ltd.



Preparation of NIPMs by the PCA Process Initially, CS nanoparticles were prepared by a simple ionic gelatin method following the reported procedure.21, 39 siRNA (1224 µg, optical density (OD) = 2.0) was dissolved in 250 µL of water containing sodium tripolyphosphate (1 mg/mL) and then mixed with 500 µL of CS solution (2 mg/mL) by gentle vortexing for 1 min. Further, the prepared CS nanoparticles were loaded with the PLLA PMs by following the reported procedure of PCA process.29,

40

PLLA (306.6 mg) and PF-127 (153.3 mg) were

dissolved in DCM (20 mL), and the emulsifier and pore-forming agent menthol (306.6 mg) was then added to the above solution to achieve an oil phase. GLP-1 (0.25 mg) was dissolved in 200 µL of deionized water and added to the oil phase. Next, 2.0 mL of deionized water containing CS nanoparticles was added to the oil phase. AB (2 mL) at a concentration of 250 mg/mL acted as the water phase. The water phase was added to the oil phase under ultrasonication to obtain a homogeneous solution, and the mixture was subjected to supercritical processing. Herein, the CS nanoparticles were added to water (dispersion phase) as they could readily disperse and retain in the water phase due to hydrophilicity of CS while insolubility at neutral pH and eventually lead to the homogenous distribution over PMs. In a typical PCA process (Figure 2), the CO2 fed from the cylinder was cooled down to approximately 0 °C using a cooler, and then delivered by a high-pressure pump. Afterwards, the liquefied CO2 was heated, and then the flow rate of CO2 was regulated steadily at 40 g/min after when the operating parameters reached to the desired levels (i.e., pressure=8 MP and temperature=30 °C). In addition, the homogeneous solution was fed into the high-pressure vessel through a nozzle with an inner diameter of 0.006 inches at a flow rate of 4.0 mL/min. Next, the obtained emulsion droplets were washed with fresh CO2 to remove the residual organic solvent.


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After washing, the autoclave was gradually depressurized, and the microspheres containing AB were collected. The harvested microspheres were eventually dried under vacuum at 50 °C to decompose the AB and yield the porous structure.

Figure 2. Schematic illustration demonstrating the instrument setup of the PCA process for the production of NIPMs and the consequences of particle formation.

Physical Characterizations The surface morphology of the samples was investigated using transmission electron microscope (H-7650, TEM, Hitachi, Japan) and field emission-scanning electron microscopy (S4800 UHR FE-SEM, Hitachi, Japan). The samples were prepared by absorbing them onto the



conducting resin and then sprayed with gold under vacuum. The particle size distribution of CS nanoparticles was obtained from laser particle size analyzer (ZetaPALS, Malvern, UK). The distribution of CS nanoparticles in PMs was determined by observing the fluorescein isothiocyanate (FITC)-tagged CS nanoparticles under confocal laser scanning microscopy (CLSM, Carl Zeiss Meta LSM510, Germany). An ultramicro spectrophotometer (Nanodrop, Thermo Scientific, USA) was used to measure the loading as well as the released amounts of siRNA. Furthermore, the elemental mapping of the NIPMs was performed by energy dispersive spectroscopy (EDS) to detect and map the distribution of CS nanoparticles in PMs. Aerodynamic Properties To elucidate the practical deposition of particles into the lung, the aerodynamic properties of the PMs such as geometric mean diameter (Dg) of the particles and their size distribution were investigated by a laser particle size analyzer (ZetaPALS, Malvern, UK). The aerodynamic diameter (Da) was measured by an eight-stage Andersen Mark II cascade impactor (ACI 20-810, Thermo Scientific, USA). The capsule containing 10 mg of PLLA PMs loaded with GLP-1 were capsulated into hard gelatin capsules (size 3, referenced by the 2015 Chinese pharmacopeia)41 and placed in a dry powder inhaler device with an orifice, from which the samples were absorbed into the ACI at a flow rate of 28.3 L/min. The effective cutoff Da for each stage was: Stage 0, 9 µm; Stage 1, 5.8 µm; Stage 2, 4.7 µm; Stage 3, 3.3 µm; Stage 4, 2.1 µm; Stage 5, 1.1 µm; Stage 6, 0.65 µm; Stage 7, 0.43 µm, Stage 8, 10 µm, Da < 4.7 µm, FPF > 60%). As shown in Table 2, the Da and Dg were increased with the amount of GLP-1 increasing, but PLLA PMs at different theoretical drug loading amounts all exhibited the aerodynamic properties in the acceptable range.30, 51 To evaluate the lung deposition efficiency, we calculated the FPF of PLLA PMs, which could be correlated to the aerodynamic performance of particles in lungs. Hickey and coworkers explored the theoretical relationship between the lung deposition of particles to their FPF, which resulted in the lung deposition efficiency of 1.4±0.6% and 20.9±3.6% at a respirable FPF of 30% and 50%, respectively. Moreover, the FPF of NIPMs at different loading amounts of GLP-1 was higher than 60%. Therefore, these PLLA PMs would result in a promising performance in lung deposition.



Figure 4a illustrates the drug loading and encapsulation efficiency of NIPMs prepared by the PCA process. Both the efficiencies increased with the theoretical loading amount of GLP-1. At different theoretical drug loading amount of GLP-1 (5.0, 7.5, and 10.0%), the corresponding loading efficiencies were 3.60, 6.28, and 8.80%, and their encapsulation efficiencies were 41.5, 45.4, and 56.3%, respectively (Figure 4a). It is evident that GLP-1 was encapsulated in the matrix of NIPMs and displayed a homogenous distribution. To verify the release efficiency of GLP-1 from PMs, the NIPMs were dispersed in PBS (pH=7.4) mimicking the physiological fluids, and the released amounts were measured by recording the absorbance using UV-Vis spectrophotometer. As shown in Figure 4b, the release behavior of GLP-1 from the NIPMs in samples with different theoretical drug loading amounts of GLP-1 was slow, and the released amounts in the initial 4 hours were 24.7, 26.9, 45.3%, respectively, demonstrating that the release of GLP-1 was proportional to its theoretical loading amount. The initial release of GLP-1 from NIPMs in the first 4 hours was mainly attributed to the peptide adsorbed on the surface of PMs through weakly bound electrostatic interactions. Further, the release lasted longer in a sustained fashion for more than 36 hours in all the samples due to its encapsulation in the polymeric framework of PMs, and dispersed nanoparticles would also avoid the burst release of GLP-1 from the PLLA framework.52-56 It is evident that NIPMs prepared by the PCA process would provide a significant potential in extending the release and shelf-life of GLP-1 in vivo, after being inhaled into the lungs and then engaging with the systemic circulation. These observations are in agreement with the existing reports in the literature, demonstrating that the biodegradable nano-micro carrier systems exhibited high entrapment of therapeutic molecules (~90%) and promising in vitro sustained release profiles.57


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Figure 4. (a) GLP-1 loading and encapsulation efficiencies in NIPMs at various theoretical loading amounts. (b) Cumulative GLP-1 release characteristics of NIPMs in PBS (pH=7.4).

In general, the particulate matter when delivered through inhalers induces lung injury and yields undesired release of inflammatory mediators and cell constituents like enzymes due to their irregular architecture and rough surface.58-59 The levels of various biochemical substances indicate the extent of damage to the cytomembrane in the lungs. More often, the ALP and LDH are the important biochemical markers that are predominantly considered to predict the safety of lung tissues after inhaling the powdered formulations since these are specific markers of cytotoxicity and explicitly indicate the damaged cells. For biosafety assessment of NIPMs the most preferred model drug is LPS, which efficiently causes the acute lung injury was chosen as positive control, accompanied by significantly increased ALP and LDH level in BALF.60 To ensure the safety of the inhalable NIPMs produced by the SCF process, the delivery device that was highly suitable and compatible for pulmonary delivery without affecting the particle properties as it does not exhibit any serious mechanical shear or aberrations during the administration process. The effects of naked NIPMs were examined by administering them to the



lungs of SD rats along with the control experiments, i.e., negative control, air treatment, and positive control groups. During the treatment, no significant change in the eventual weight of SD rats was observed, demonstrating that the treatment at the recommended dose of NIPMs was well-tolerated. As shown in Figure 5a, the results demonstrated that the ALP levels in the BALF of SD rats inhaled with NIPMs and air treatment were slightly higher (1.23-fold and 1.18-fold respectively) than that of the untreated group. The minor change in the levels of biomarkers might be caused by the air pressure and the turbulent movement of microspheres indicating acute lung injury during the pulmonary administration. In addition, the levels of LDH in the BALF of NIPMs treated group were in the acceptable range and exhibited no significant difference with the negative control group in both the cases (Figure 5b), demonstrating that no substantial damage happened to the tissues.61 To this end, the positive control, LPS had induced a great extent of damage to the lung tissue yielding significant ALP (1.81-fold ) and LDH (4.99-fold) levels in the BALF than that of the untreated group.62-63 Comparatively, no substantial inflammation resulting in the significantly lower levels of ALP and LDH in the BALF of NIPMs treatment group could be related to the biocompatibility and biodegradability of particles in the lungs. Further, the effect of NIPMs on the damage of lung tissue was confirmed by H&E staining of lung sections along with control experiments. The results of lung tissues of SD rats showed no significant changes in the epithelium (Figure 5c), indicating the potential compatibility of NIPMs, which allows them to be applied as a safety carrier for diverse biomedical applications.


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Figure 5. Biosafety assessment of NIPMs in vivo. The levels representing the quantitative assessment of (a) ALP, and (b) LDH in the BALF of lungs after administering NIPMs through the pulmonary route. * represents statistical significance, P

Supercritical Fluid-Assisted Decoration of Nanoparticles on Porous

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