Micromechanical Punching: a Versatile Method for Non-Spherical Microparticle Fabrication

Communication Micromechanical Punching: A Versatile Method for Non-Spherical Microparticle Fabrication

Ritika Singh Petersen 1,2,*, Anja Boisen 1,3 and Stephan Sylvest Keller 1,2

1 DNRF and Villum Fonden Center for Intelligent Drug Delivery and Sensing Using Microcontainers and Nanomechanics, IDUN, DTU Health Technology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; [email protected] (A.B.); [email protected] (S.S.K.) 2 National Centre of Nano Fabrication and Characterization, DTU Nanolab, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark 3 Department of Health Technology, DTU Health Tech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark * Correspondence: [email protected]; Tel.: +45-45258109

Abstract: Microparticles are ubiquitous in applications ranging from electronics and drug delivery to cosmetics and food. Conventionally, non-spherical microparticles in various materials with specific shapes, sizes, and physicochemical properties have been fabricated using cleanroom-free lithography techniques such as soft lithography and its high-resolution version particle replication in non-wetting template (PRINT). These methods process the particle material in its liquid/semi-liquid state by deformable molds, limiting the materials from which the particles and the molds can be fabricated. In this study, the microparticle material is exploited as a sheet placed on a deformable substrate, punched by a robust mold. Drawing inspiration from the macro-manufacturing technique of punching metallic sheets, Micromechanical Punching (MMP) is a high-throughput technique for fabrication of non-spherical microparticles. MMP allows production of microparticles from prepatterned, porous, and fibrous films, constituting thermoplastics and thermosetting polymers. As an illustration of application of MMP in drug delivery, flat, microdisk-shaped Furosemide embedded poly(lactic-co-glycolic acid) microparticles are fabricated and Furosemide release is observed. Thus, it is shown in the paper Citation: Petersen, R.S.; Boisen, A.; that Micromechanical punching has potential to make micro/nanofabrication more accessible to the Keller, S.S. Micromechanical research and industrial communities active in applications that require engineered particles. Punching: A Versatile Method for Non-Spherical Microparticle Keywords: non-spherical microparticle; soft lithography; drug delivery; punching Fabrication. Polymers 2021, 13, 83. https://doi.org/10.3390/polym1301 0083 1. Introduction Received: 27 November 2020 Accepted: 23 December 2020 In order to influence the biodistribution of drug-loaded micro- and nanoparticles in Published: 28 December 2020 pharmaceutics and the targeting, release profile, and bioavailability of the drug, the ability to precisely manipulate size, shape, and surface properties of the particles is important [1,2]. Publisher’s Note: MDPI stays neu- Similarly, the composition, size and shape of a metallic nanoparticle dictate the resonant tral with regard to jurisdictional claims frequency of the electron oscillation in localized surface plasmonics. This phenomenon is in published maps and institutional used in detecting analytes bound to molecules immobilized on the metallic particle, which affiliations. is applied in biosensors, immunoassays, and spectroscopy [3,4]. Thus, novel applications in drug delivery, tissue engineering, and biosensing are pushing towards novel fabrication techniques. These techniques can produce specifically designed particles, with additional functionalities and sensitive cargos such as drugs or biomolecules [5,6]. Copyright: © 2020 by the authors. Li- Non-spherical microparticles with precisely controlled physical and chemical proper- censee MDPI, Basel, Switzerland. This ties have been conventionally fabricated by using. article is an open access article distributed Microelectromechanical systems (MEMS) and semiconductor technologies such as under the terms and conditions of the photolithography and dry etching [7,8]. However, these processes are expensive, have low Creative Commons Attribution (CC BY) license (https://creativecommons.org/ throughput, and can sometimes degrade the cargo in the particle susceptible to UV light or licenses/by/4.0/). high processing temperatures. Soft lithography is an alternative approach for non-spherical

Polymers 2021, 13, 83. https://doi.org/10.3390/polym13010083 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 83 2 of 9

microparticle fabrication [9]. It is an umbrella term for various prototyping methods such as microcontact printing, replica molding, microtransfer molding, micromolding in cap- illary (MIMIC), solvent-assisted Micromolding, and more recently particle replication in non-wetting template (PRINT) [10,11]. The common feature for all the aforementioned processes is the application of a “soft” deformable mold made of materials such as poly- dimethylsiloxane (PDMS) for printing, embossing, or molding of the microparticle material. However, such a mold is susceptible to damage already after a few runs of processing. In order to address this limitation for microparticle fabrication, hot punching was devel- oped [12]. In this method, the particle material was prepared on a deformable substrate and patterned by a durable Ni mold instead of a deformable mold [12]. This allowed for large-scale fabrication of high aspect ratio reservoir-based particles for oral drug delivery, declared as microcontainers, in biodegradable and biocompatible polymers [13,14]. How- ever, the application of heat during hot punching of microparticles for drug delivery can lead to degradation of sensitive cargo included in the microparticles such as biomolecule drugs. Furthermore, heating typically implies longer processing time and requires more advanced microfabrication equipment. Another common feature of soft lithography and hot punching methods is the ma- nipulation of some form of ink or liquid/semi-liquid precursor solution as material for microparticle fabrication. Alternatively, the microparticle material can be a solid sheet that can be deformed and cut to fabricate microparticles with desired structures and dimensions. This new perspective of handling the microparticle layer as a deformable sheet is inspired by a high throughput macromanufacturing technique called punching or blanking that has been used for metal sheet forming for hundreds of years. Blanking and punching are shearing processes in which a punch and die are used to modify webs [15]. Inspired by the punching process, we introduce Micromechanical Punching (MMP) as a simple, benign, and fast process for fabrication of microparticles. Here, we demonstrate the concept and potential of MMP by fabrication of a variety of microparticles with different materials that can potentially be used for a wide array of applications. Finally, as an illustrative example, we show the application of MMP to produce poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with Furosemide (Furo) for oral drug delivery.

2. Materials and Methods 2.1. Materials Dichloromethane (DCM) (anhydrous >99.8%), Poly-e-caprolactone (PCL) pellets (average Mw~100,000), Poly(acrylic acid) (PAA) solution (average Mw~100,000, 35 wt.% in H2O), Acetone (>99.5% purity), and gelatin powder were obtained from Sigma-Aldrich (Copenhagen, Denmark). Phosphate buffer saline (PBS) tablets and Furosemide (≥98% purity) acquired from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water was obtained from MilliQ Integral Water Purication System for Ultrapure Water, manufactured by Merck Mil- lipore (Burlington, MA, USA). Poly(Lactic-co-glycolic acid) (PLGA) 50:50 (acid end cap, Mn 85,000–100,000 g/mol) was obtained from Akina, IN, US. Poly(L-lactic acid) (PLLA) 2003D grade is purchased from Natureworks (Minnetonka, MN, USA), with Mw = 126,000 Da, de- termined by size exclusion chromatography measurements. Polytetraﬂuoroethylene sheets (PTFE sheet of Fluoroplast, opaque) were bought from RS Components A/S. Cellulose weighing paper was acquired from VWR European. Co-extruded polyethylene (PE) and polypropylene (PP) ﬁlms were obtained by Inmold A/S.

2.2. Scanning Electron Microscopy and Stylus Profilometry A TM3030Plus (Hitachi, Japan) tabletop SEM was used for scanning electron microscopy (SEM). The micrographs were obtained at an operating voltage of 15 KV in charge reduction mode using mixed secondary electrons (SE) and backscattered electron (BSE) detector signal. The thicknesses of the PLGA-Furo films were measured using KLA-Tencor Alpha Step IQ stylus profilometer (Milpitas, CA, USA). The thickness of the film was measured by Polymers 2021, 13, 83 3 of 9

mechanically removing a part of the coated area to expose the substrate beneath. Then the thickness was measured by doing step height analysis. An average of ﬁve thickness measurements on the PLGA-Furo ﬁlm is reported here.

2.3. Film Production Using Blade Coating For blade coating, first, a homogenous solution of polymer or polymer–drug was prepared. The homogenous solution of biocompatible and biodegradable polymers like PCL, PLLA, and PLGA was prepared as 10 wt.% in DCM solvent. The water soluble polymers like gelatin and PAA were dissolved as 5 wt.% concentration solutions. These solutions were dispensed on an Erichson blade coating machine, and the blade height was adjusted as per the thickness required. PCL, PLLA, and PLGA solutions were coated with blade height ≈ 0.5 mm, while gelatin and PAA solutions were coated with blade height ≈ 0.1 mm. The blade was then moved at a slow speed of 0.5 m/min. The film was left overnight for solvent evaporation. The films were either directly prepared on the deformable substrate or were coated on a PTFE sheet and peeled later. In order to prepare PLGA-Furo films for the in vitro release of Furosemide from PLGA, a homogenous solution of 10 wt.%, consisting of 1:5 Furosemide to PLGA in acetone and DCM, was prepared. The solution was blade coated on a PTFE sheet and left overnight at room temperature for solvent evaporation. This resulted in a 40 ± 4 µm thick PLGA-Furo film. The film was peeled from the PTFE sheet and transferred to a PP sheet.

2.4. Preparation of Formulation: Capsule Loading First the lid of the rat capsule is weighed without any microparticles. Then, the cap of the capsule is taken off and the capless capsule is placed in a capsule holder. The particles lying on the deformable substrate were scraped off by a lab knife and gently nudged in a funnel that sits on the top of a stainless steel holder. The holder is specifically designed for the capsule in terms of shape and size. A funnel is then placed on this holder and then the microparticles are scraped off the deformable substrate with the help of a lab knife. The scraped microparticles are allowed to fall on the funnel in order to fill the capsule. Sometimes the microparticles clogging the funnel hole are gently nudged in the capsule by a piston. During and after the filling of the capsule, it is weighed with the cap on. Approximately 0.5 mg of particles was weighed in the capsules for the in vitro release studies.

2.5. In Vitro Release of Furosemide from PLGA Matrix In vitro drug release was measured in dissolution studies performed with a microdis- solution profiler [16]. The PLGA-Furo particles were filled in the rat gelatin capsules and placed in the glass vials filled with 10 mL of phosphate buffer (pH 7.5) solution (PBS). The gelatin capsules are predominantly dissolved within few seconds of being placed in the PBS media. Experiments were performed at 37 ◦C using a stirring rate of 200 rpm. The path length of UV probes was chosen as 20 mm on the µDiss profiler (Pion, United Kingdom). Standard curve for the calibration of the channels was performed prior to the release experiments. For the standard curve, Furosemide in milliQ (pH = 10) stock solution was prepared. A fixed amount of this solution was added to 10 mL of PBS. The defined concentration was measured by detecting the UV absorbance from the added analyte in the stock solution. After the calibration of the channels, the actual experiment was performed with the capsules by scanning the samples by the in situ UV probes every 30 s with a run time of 7 h. The release experiments are expressed as normalized (to the maximum drug released after 7 h) drug release over time. The experiments were performed in 6 replicates from 3 independent productions within a week of film and particle formation.

2.6. Micromechanical Punching (MMP) The conventional punching or blanking process includes three basic steps illustrated in Figure1A: (1) A metal sheet is positioned on an inﬂexible die. (2) The punch and the Polymers 2021, 13, x FOR PEER REVIEW 4 of 9

ments were performed in 6 replicates from 3 independent productions within a week of film and particle formation.

Polymers 2021, 13, 83 2.6. Micromechanical Punching (MMP) 4 of 9 The conventional punching or blanking process includes three basic steps illustrated in Figure 1A: (1) A metal sheet is positioned on an inflexible die. (2) The punch and the metalmetal sheet sheet are are brought brought into into contact contact by by the the application application of a high high force. force. The The penetration penetration of of thethe punch punch into into th thee voids voids of of the the die die results results in in build build-up-up of of stresses in the metal sheet with thethe maximum stress stress locatedlocated at at the the edges edges of of the the punch. punch. When When the the maximum maximum stress stress exceeds ex- ceedsthe ultimate the ultimate tensile tensile strength strength of the metal,of the themetal, metal the sheet metal below sheet the below punch the is punch cut free is from cut freethe surroundingfrom the surrounding metal. (3) metal. Finally, (3 the) Finally, punch the and punch the die and are the separated die are separated [15]. [15].

Conventional Punching Micro- Mechanical Punching A B

ii iii iv C.i

Figure 1. (A) ConventionalFigure punching 1. (A vs.) Conventional (B) micromechanical punching punching; vs. (B) micromechanical (C) do-it-yourself punching; punching (C using) do-it Playdough-yourself ® ® ® and Lego® block showing thepunching basic steps using of Playdough the MMP process: and Lego (i) sample block showing preparation, the basic (ii) MMP, steps (iii) of the demolding MMP process: the mold (i) sample preparation, (ii) MMP, (iii) demolding the mold from the microparticle layer, and (iv) mi- from the microparticle layer, and (iv) microparticles obtained on the deformable substrate after peeling off the surrounding croparticles obtained on the deformable substrate after peeling off the surrounding punched film punched film (Green: Hard mold; red: Microparticle layer; blue: Deformable substrate). (Green: Hard mold; red: Microparticle layer; blue: Deformable substrate). The two significant differences of the proposed MMP process compared to the con- ventionalThe two punching significant technique differences are the of utilization the proposed of a deformable MMP process substrate compared and a hollow to the conventionalpunch. In MMP, punching the microparticle technique layerare the is placed utilization on a of deformable a deformable substrate substrate in the and form a hollowof a solid punch. material In MMP, sheet. the This microparticle flexible stack layer of the is placed substrate on a and deformable the microparticle substrate layer in the is formpunched of a bysolid a hard material but hollowsheet. This punch flexible or mold. stack During of the MMP,substrate the cuttingand the ormicroparticle indentation layerof the is microparticle punched by layera hard by but the hollow hollow punch moldand or mold. the final During punching MMP of, the the cutting microparticle or in- dentationlayer occur of in the a single microparticle step, leading layer to by the the fabrication hollow ofmold microparticles and the final of controlledpunching shapes,of the microparticlesizes, and surface layer topography occur in a single (Figure step,1B,C). leading to the fabrication of microparticles of controlledThe MMP shapes, process sizes, startsand surface with thetopography preparation (Figure of the 1B deformable,C). substrate, the mi- croparticleThe MMP layer, process and the starts hard with mold the (Figure preparation1(Ci)). of For the the deformable deformable substrate, substrate, the com- mi- croparticlepression molded layer, orand extruded the hard polymer mold ( sheets,Figure solution-casted1Ci). For the deformable cross-linked substrate, PDMS layers, com- pressioncommercially molded available or extruded aluminum polymer (Al) sheets,sheets, orsolution polymer-casted films cross prepared-linked from PDMS solutions layers, by commerciallyspin coating, bladeavailable coating, aluminum or spray (Al) coating sheets are, or suitable polymer [17 films]. As prepared high pressure from issolutions applied byduring spin mechanicalcoating, blade punching, coating,a or robust spray deformable coating are substrate suitable [1 is7 important.]. As high pressure For example, is ap- a pliedPDMS during substrate mechanical succumbs punching, under 5 bars a robust platen deformable pressure while substrate a polyethylene is important. (PE) substrate For example,can tolerate a PDMS platen substrate pressures succumbs >20 bars. under The microparticle5 bars platen layerpressure can while be materials, a polyethylene ranging (PE)from subs polymerstrate can and tolerate gels to platen metals. pressures At room >20 temperature bars. The microparticle any material layer which can is notbe ma- too terials,ductile ranging as poly- ε from-caprolactone polymers and and too gels brittle to metals. as ceramics At room can temperature be mechanically any punched. material whichHowever, is not in principle, too ductile ductile as poly materials-ε-caprolactone can be frozen and and too brittle brittle materials as ceramics can be can processed be me- chanicalat elevatedly punched. temperatures However, for successful in principle, punching. ductile The materials microparticle can be layer frozen can and be directly brittle materialsdeposited can on the be deformable processed at substrate elevated by temperatures above mentioned for successfulcoating techniques punching. or prepared The mi- croparticleseparately layer and then can transferred be directly todeposited the deformable on the deformable substrate before substrate MMP. by Finally, above MMPmen- tionedrequires coa ating robust techniques mold that or prepared does not separately succumb underand then the transferred pressure applied. to the deformable The mold substrateconsists of before large MMP. arrays Finally, of protruding MMP requires walls around a robust a cavity mold of that desired does sizenot andsuccumb shape, un- as dershown the pressure in Figure applied.1(B,Ci). The In general, mold consists the mold of large material arrays should of protruding have a higher walls hardnessaround a than the other materials in the stack. Molds with sharp protrusions are required for easy punching, while mold features with low roughness and slightly positive sidewall angles are necessary for easy demolding after the MMP process. The mold protrusions should be higher than the thickness of the material layer to allow for complete punching. After preparation of the mold, the material layer and the deformable substrate are prepared. The complete stack is placed in a parallel plate (P2P) embosser. High pressure Polymers 2021, 13, 83 5 of 9

is applied on the stack in order to punch the microparticle layer (Figure1(Cii)). After the punching process, the mold is separated from the microparticle layer (Figure1(Ciii)). The leftover ﬁlm in between the punched particles is peeled from the substrate (Figure1(Ciii)) and the microparticles either remain on the deformable substrate (Figure1(Civ,D)) or are transferred to the mold. In the latter case, another step of bonding or particle transfer to a desired substrate is performed. The micromechanical punching was performed using the hot embosser (Collin® Press, Ebersberg, Germany, 300 SV). The particles were generally punched at pressures >20 bars for 1 min.

3. Results and Discussion 3.1. Fabrication of Microparticles with Various Materials, Shapes and Sizes For the demonstration of the MMP process, a Ni mold with protruding walls, as shown in Figure2A, was fabricated by using a dry etching, electroplating, and molding (DEEMO) process [18]. A molecular layer of perfluorodecyltrichlorosilane (FDTS) was deposited on the Ni mold using molecular vapor deposition (MVD). This improves the separation of the mold and the punched polymer microparticles by reducing the adhesion forces between the two materials. MMP was performed at platen pressures ≥20 bars for 1 min. Figure2A–C shows Ni molds used in MMP with protrusions of square, circular, and hexagonal shapes, respectively. The diagonal of the square microstructures on the mold has a length 200 µm. The height of the protrusions is 70 µm and the thickness of the walls is 20 µm. The diameter of the circular microstructures on the mold is 260 µm. The height of the protrusions is same as the square microstructures, that is, 70 µm while the wall thickness is 40 µm. The hexagonal Ni stamp shown in the Figure2C has protrusions of height = 20 µm, diagonal length = 20 µm, and wall thickness = 4 µm. As an initial proof of concept, MMP was performed on uniform single material microparticle layers such as metal sheets and polymer films. These films had thicknesses measured in the range of 10 µm to 60 µm. To evaluate punching of biodegradable and biocompatible polymers, 14.7 wt.% of Poly-l-lactic acid (PLLA) solution in dichloromethane (DCM) was blade-coated on a polytetrafluoroethylene (PTFE) sheet, peeled off the PTFE surface and then placed on a PE substrate layer for punching. MMP was performed and square shaped PLLA particles were obtained on the deformable PE layer as shown in Figure2D. Figure2E shows a punched and dried gelatin layer lying on a carbon tape. The gelatin layer was obtained by blade coating of a 20 wt.% aqueous collagen solution on an oxygen plasma treated PP sheet and drying the film overnight. The gelatin layer was punched, after which the gelatin particles remained attached to the Ni mold and were finally harvested on a carbon tape. In order to fabricate smaller microstructures, an aqueous solution of 15 wt.% Poly Acrylic Acid (PAA) was coated on a plasma treated PP sheet using blade coating. The wet film was left overnight, which was later on punched by Ni mold having hexagonal protrusions of height = 20 µm, diagonal length = 20 µm, and wall thickness = 4 µm. The punching was performed under a higher platen pressure of 40 bars for 1 min to accommodate for the smaller sizes. PAA hexagonal microparticles were obtained on the PE film after demolding as shown in Figure2F. Polymers 2021, 13, 83 6 of 9 Polymers 2021, 13, x FOR PEER REVIEW 6 of 9

A B C

Ni Ni

Ni 200µm 400µm 100µm

D E F

PLLA GELATIN PAA 100µm 400µm 100µm 10µm 300µm 100µm

Figure 2. (A–C) SEM micrographsFigure 2. showing (A–C) SEM the Nimicrographs molds used showing for MMP; the ( DNi) squaremolds used microparticles for MMP; punched(D) square from microparticles poly-l- lactic acid polymer ﬁlm; (E)punched circular microparticlesfrom poly-l-lactic punched acid polymer from gelatin film; ﬁlm; (E) circular and (F) microparticles hexagonal particles punched in water from soluble gelatin film; and (F) hexagonal particles in water soluble polymer PAA with order magnitude smaller polymer PAA with order magnitude smaller sizes. sizes.

3.2.3.2. Fabrication Fabrication of of Microparticles Microparticles from from Pre Pre-Textured-Textured Films: Naturally Naturally Occurring or Artificially PatternedArtificially Patterned FabricationFabrication of microparticles fromfrom polymerpolymer films films with with nanostructures, nanostructures, surface surface texture, tex- tureor those, or those consisting consisting of porous of porous materials materials with processes with processes such as such soft lithography as soft lithography or PRINT or is PRINTchallenging. is challenging MMP is ideal. MMP for producing is ideal microparticles for producing from microparticles such pre-patterned from films. such In preFigure-patterned3A, microparticles films. In Figure punched 3A, outmicroparticles of cellulose punched paper on out extruded of cellulose polypropylene paper on (PP) ex- trudedfilm as polypropylene deformable substrate (PP) film are shown.as deformable The close-up substrate of the are particles shown. shows The close that the-up fibrousof the particlesstructure shows of the that paper the wasfibro maintainedus structure after of the the paper punching was maintained process. To after demonstrate the punching that process.this process To demonstrate can be further that applied this toprocess fabricate can particles be further from applied natural to materials fabricate displaying particles frompatterns, natural a dried materials beech displaying hedge leaf patterns, (Fagus a sylvatica dried beech) was hedge punched leaf while(Fagus lyingsylvatica on) awas PP punchedsheet. Figure while3 lyiB depictsng on a PP the sheet. retention Figure of 3 theB depicts leaf patterns the retention on the of fabricated the leaf patterns particles. on theFigure fabricated3(Ci,Cii) particles. shows hexagonal-in-hexagonalFigure 3(Ci,Cii) shows hexagonal hierarchical-in-hexagonal particles. hierarchical These particles particles.were fabricated These particles from a were poly(lactic-co-glycolic fabricated from a acid) poly(lactic (PLGA)-co- filmglycolic which acid) was (PLGA) obtained film by whichblade coatingwas obtained 10 wt.% by PLGA blade solutioncoating 10 in DCMwt.% PLGA on a PTFE solution sheet. in TheDCM coated on a solutionPTFE sheet. was ◦ Thedried coated overnight. solution The was resulting dried overnight. film was The first resulting hot embossed film was at 80firstC hot for embossed 5 min by at a 80 Ni µ °Cmold for with5 min honeycomb by a Ni mold hexagonal with honeycomb structures hexagonal with a feature structures size of with 2 m.a feature Afterward, size of the 2 µm.patterned Afterward, film wasthe patterned removed fromfilm was the PTFEremoved sheet from and the placed PTFE on sheet a PE and film. placed This on stack a PE of film.PLGA This and stack PEfilms of PLGA was mechanicallyand PE films punchedwas mechanically by the Ni punched mold with by hexagonal the Ni mold features. with These hexagons have diagonals of 250 µm in length, height of walls = 70 µm and wall hexagonal features. These hexagons have diagonals of 250 µm in length, height of walls = thickness = 20 µm. Punching of the prepatterned film with this mold results in particles 70 µm and wall thickness = 20 µm. Punching of the prepatterned film with this mold re- with hierarchical structures. sults in particles with hierarchical structures.

Polymers 2021, 13, 83 7 of 9 Polymers 2021, 13, x FOR PEER REVIEW 7 of 9

B A

CELLULOSE CELLULOSE LEAF 100µm LEAF 100µm

300µm 400µm

C.i C.ii

PLGA

PLGA 300µm 50µm

Figure 3. Mechanical punching:Figure (A 3.) microparticles Mechanical punching: from cellulose (A) micropart paper showingicles from the cellulose ﬁbrous structure paper showing of the microparticle, the fibrous (B) microparticles from driedstructure fall leaf of showing the microparticle the leaf patterns,, (B) microparticles and (C) hexagonal from dried hierarchal fall leaf microparticles. showing the leaf patterns, and (C) hexagonal hierarchal microparticles.

3.3. Micromechanical Micromechanical Punching of Drug Drug-Loaded-Loaded Microparticles One o off the major advantages of MMP lies in the production of regular and uniform particles as comparedcompared toto thethe conventional conventional methods methods for for the the production production of of spherical spherical particles parti- cleswhich which usually usually have have a broad a broad particle particle size distribution. size distribution. Moreover, Moreover, MMP leadsMMP to leads the fabrica- to the fabriction ofation flat asymmetric of flat asymmetric particles particles that can that resist can the resist peristaltic the peristaltic flow of intestinal flow of fluids intestinal and fluidsallow betterand allow contact better between contact the between mucosa walls.the mucosa This leads walls. to This better leads adhesion to better and adhesion retention andof the retention asymmetric of the particles asymmetric and hence,particles higher and bioavailabilityhence, higher bioavailability of the drug [19 of]. Here,the drug flat [1PLGA-Furo9]. Here, flat microparticles PLGA-Furo are microparticles fabricated using are MMP. fabricated Furosemide using is MMP. a loop Furosemide diuretic usually is a loopfound diuretic in a stable usually crystalline found form in a stablewith very crystalline low solubility form with in aqueous very low media solubility at room in aqueoustemperature. media It hasat room been temperature. previously proven It has thatbeen drugs previously dispersed proven in solid that polymer drugs dispe matricesrsed incan solid stay polymer in stable matrices amorphous can forms, stay in increasing stable amorphous the bioavailability forms, increasing of the drug the [ 20bioavaila-]. There- bilityfore, Furosemideof the drug is[20 embedded]. Therefore, in PLGA Furosemide polymer is matrixembedded as microdisk-shaped in PLGA polymer PLGA-Furo matrix as microdiskmicroparticles.-shaped PLGA-Furo microparticles. For the the fabrication fabrication of ofPCL-Furo PCL-Furo flat microparticles, flat microparticles, the PLGA-Furo the PLGA film-Furo was film punched was punchedwith 20 bars with for 20 1 min.bars for Figure 1 min.2C depictsFigure the2C Nidepicts mold the and Ni Figure mold4 Aand illustrates Figure 4 theA illustrates punched thePLGA-Furo punched particles PLGA-Furo on the particles PP substrate. on the FigurePP substrate.4B shows Figure that the4B shows punched that microparticles the punched microparticlesremain porous remain after the porous MMP after process. the MMP process. After the drugdrug-loaded-loaded microparticles were fabricated, they they were filledfilled in gelatin capsules for for release studies. Figure Figure 44CC shows a dissolutiondissolution profileprofile wherewhere thethe drugdrug con-con- centrations are are normalized with with respect respect to the maximum drug concentration after after 7 h of dissolution study. study. The The drug drug release release occurs occurs primarily primarily through through diffusion diffusion as as 5 5–7–7 h is not long enough to lead to the degradation of PLGA in PBS buffer (pH = 7.5) [14]. The drug long enough to lead to the degradation of PLGA in PBS buffer (pH = 7.5) [14]. The drug release profile in Figure4C represents an initial burst release where approximately 50% of release profile in Figure 4C represents an initial burst release where approximately 50% drug is release within first 5 min. The high burst release can be attributed to a Furosemide of drug is release within first 5 min. The high burst release can be attributed to a Furo- rich surface and the amorphous form of Furosemide in the PLGA-Furo microparticles. semide rich surface and the amorphous form of Furosemide in the PLGA-Furo micro- During the PLGA-Furo film formation, the solvent evaporation of film lead to Furosemide particles. During the PLGA-Furo film formation, the solvent evaporation of film lead to

Polymers 2021, 13, 83 8 of 9 Polymers 2021, 13, x FOR PEER REVIEW 8 of 9

getting distributed closely to the surface of the film [21]. As there is no further movement of FurosemideFurosemide getting in the solidifieddistributed film closely during to the the surface MMP of process, the film Furosemide-rich [21]. As there is no surface fur- in thether microparticles movement is of obtained. Furosemide Moreover, in the solidified Furosemide film is during quenched the MMP in its process, amorphous Furo- form semide-rich surface in the microparticles is obtained. Moreover, Furosemide is quenched due to the rapid evaporation of low boiling point solvent, Dichloromethane [21]. With in its amorphous form due to the rapid evaporation of low boiling point solvent, Di- no heat applied during the MMP process, Furosemide dispersed in the matrix of PLGA chloromethane [21]. With no heat applied during the MMP process, Furosemide dis- microparticlespersed in the continues matrix of toPLGA stay inmicroparticles its amorphous continues form. to After stay the in initialits amorphous burst release, form. the remainingAfter the drug initial release burst fromrelease, the the deeper remaining layers drug of the release polymer from matrix the deeper becomes layers slow. of the These experimentspolymer matrix demonstrate becomes that slow. the These microparticles experiments fabricated demonstrate by that MMP the can microparticles be utilized in multiparticulatefabricated by MMP drug can delivery be utilized systems in multiparticulate [22]. drug delivery systems [22].

A B.(i) Before MMP C 1.2

5µm 0.8

0.6

(ii) After MMP 0.4 Drug release

0.2

PLGA-Furo concentrationNormalized

600µm 100µm 5µm 0 0 2 Time (hr) 4 6

Figure 4. PLGA-Furo microparticleFigure fabrication 4. PLGA- andFuro characterization: microparticle fabrication (A) PLGA-Furo and characterization: microparticles (A) lying PLGA on-Furo the PPmicroparticles substrate lying on the PP substrate after the MMP process; (B) porosity of the blade coated PLGA-Furo films after the MMP process; (B) porositybefore of (i the) and blade after coated MMP PLGA-Furo(ii) showing that ﬁlms the before film remains (i) and afterporous MMP after (ii) the showing punching that process; the ﬁlm and remains porous after the punching(C) in process; vitro release and ( ofC) Furosemide in vitro release from ofthe Furosemide PLGA microparticles. from the PLGA microparticles.

4. Conclusions 4. Conclusions In this paper, the Micromechanical Punching (MMP) process is introduced for the fabricIn thisation paper, of microparticles. the Micromechanical Microparticles Punching of various (MMP) shapes process and sizes, is introduced made up of forbi- the fabricationodegradable of microparticles.and biocompatible Microparticles polymers like PCL, of various PLGA, shapesand PLLA and and sizes, water made-soluble up of biodegradablepolymers like and gelatin biocompatible and PAA, have polymers been successfully like PCL, PLGA, fabricated and PLLAusing MMP. and water-soluble It is also polymersdemonstrated like gelatin that MMP and can PAA, be haveused for been punching successfully prepatterned, fabricated fibrous using or porous MMP. Itfilms isalso demonstratedto successfully that fabricate MMP can hierarchal/patterned, be used for punching fibrous prepatterned,, or porous fibrousmicroparticles, or porous respec- films to successfullytively. Some fabricate of the examples hierarchal/patterned, shown in the paper fibrous, are orpunching porous of microparticles, fibrous cellulose respectively. sheet, SomeFagus of sylvatica the examples leaf and shown hexagon in- the patterned paper PLGA are punching films. Finally, of fibrous using celluloseMMP, Furosemide sheet, Fagus sylvatica(Furo)leaf-containing and hexagon- PLGA microparticlespatterned PLGA are films. fabricated Finally, with using uniform MMP, size Furosemide and shape. (Furo)- In containingvitro release PLGA studies microparticles are conducted are fabricated on these PLGA with uniform-Furo particles. size and Successful shape. In relea vitroserelease of studiesFurosemide are conducted from PLGA on these matrix PLGA-Furo is shown depicting particles. the Successful viability releaseof using of MMP Furosemide for drug from PLGAdelivery matrix applications. is shown depicting the viability of using MMP for drug delivery applications. MMP is a low cost, single-step process that can be scaled up to roll-to-roll produc- MMP is a low cost, single-step process that can be scaled up to roll-to-roll production, tion, which in principle can be completely solvent-free. In future, it would be interesting which in principle can be completely solvent-free. In future, it would be interesting to to develop this technology to fabricate nanoparticles that can be applied for future ap- develop this technology to fabricate nanoparticles that can be applied for future applica- plications in biotechnology and photonics. Investigation into the effect of pressure on tionsthin in film biotechnology materials, 2D and materials photonics., and Investigationsensitive cargos into embedded the effect in of the pressure films will on be thin in- film materials,teresting 2D as materials,well as required and sensitive for applications cargos embeddedlike drug delivery in the filmsand tissue willbe engineering. interesting as wellMicromechanical as required for applicationsPunching has likepotential drug deliveryto obtain andwidespread tissue engineering. application in Micromechani- academic callaboratories Punching has and potential industrial to scale obtain manufactur widespreading around application the world in academic due to the laboratories simplicity of and industrialits execution scale on manufacturing practically any around conceivable the worldmaterial due that to can the be simplicity acquired as of a its sheet execution or can on practicallybe applied any on conceivable a substrate film. material that can be acquired as a sheet or can be applied on a substrate film. Author Contributions: Conceptualization, R.S.P.; methodology, R.S.P.; software, R.S.P.; valida- Authortion, Contributions:R.S.P., formal analysis,Conceptualization, R.S.P.; investigation, R.S.P.; R.S.P., methodology, resources, R.S.P.;S.S.K. and software, A.B.; data R.S.P.; curation, validation, R.S.P.,R.S.P.; formal writing analysis,—original R.S.P.; draft investigation, preparation, R.S.P.; R.S.P., writing resources,—review S.S.K. and and editing, A.B.; R.S.P., data curation,S.S.K., and R.S.P.; writing—original draft preparation, R.S.P.; writing—review and editing, R.S.P., S.S.K., and A.B.; visualization, R.S.P.; supervision, S.S.K. and A.B. All authors have read and agreed to the published version of the manuscript. Polymers 2021, 13, 83 9 of 9

Funding: This work was supported by the Danish National Research Foundation (DNRF122) and Villum Foundation (Grant No. 9301), Center for intelligent drug delivery and sensing using microcontainers and nanomechanics (IDUN). Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Data is contained within the article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Banerjee, A.; Qi, J.; Gogoi, R.; Wong, J.; Mitragotri, S. Role of Nanoparticle Size, Shape and Surface Chemistry in Oral Drug Delivery. J. Control. Release 2016, 238, 176–185. [CrossRef][PubMed] 2. Christfort, J.F.; Guillot, A.J.; Zaera, A.M.; Thamdrup, L.H.; Garrigues, T.; Boisen, A.; Zór, K.; Nielsen, L.H. Cubic Microcontainers Improve In Situ Colonic Mucoadhesion and Absorption of Amoxicillin in Rats. Pharmaceutics 2020, 12, 355. [CrossRef][PubMed] 3. Jeong, H.-H.; Mark, A.G.; Alarcón-Correa, M.; Kim, I.; Oswald, P.; Lee, T.-C.; Fischer, P. Dispersion and Shape Engineered Plasmonic Nanosensors. Nat. Commun. 2016, 7, 11331. [CrossRef][PubMed] 4. Anker, J.N.; Hall, W.P.; Lyandres, O.; Shah, N.C.; Zhao, J.; Van Duyne, R.P. Biosensing with Plasmonic Nanosensors. Nanosci. Technol. 2010, 308–319. [CrossRef] 5. Mitragotri, S.; Lahann, J. Physical Approaches to Biomaterial Design. Nat. Mater. 2009, 8, 15–23. [CrossRef][PubMed] 6. Zhang, K.; Wang, S.; Zhou, C.; Cheng, L.; Gao, X.; Xie, X.; Sun, J.; Wang, H.; Weir, M.D.; Reynolds, M.A.; et al. Advanced Smart Biomaterials and Constructs for Hard Tissue Engineering and Regeneration. Bone Res. 2018, 6, 1–15. [CrossRef][PubMed] 7. Brown, A.B.D.; Smith, C.G.; Rennie, A.R. Fabricating Colloidal Particles with Photolithography and Their Interactions at an Air-Water Interface. Phys. Rev. E 2000, 62, 951–960. [CrossRef][PubMed] 8. Dendukuri, D.; Pregibon, D.C.; Collins, J.; Hatton, T.A.; Doyle, P.S. Continuous-Flow Lithography for High-Throughput Microparticle Synthesis. Nat. Mater. 2006, 5, 365–369. [CrossRef][PubMed] 9. Whitesides, G.M.; Ostuni, E.; Takayama, S.; Jiang, X.; Ingber, D.E. Soft Lithography in Biology and Biochemistry. Annu. Rev. Biomed. Eng. 2001, 3, 335–373. [CrossRef][PubMed] 10. Sen, A.K.; Raj, A.; Banerjee, U.; Iqbal, S.R. Soft Lithography, Molding, and Micromachining Techniques for Polymer Micro Devices; Microﬂuidic Electrophoresis; Humana Press: New York, NY, USA, 2019; pp. 13–54. 11. DeSimone, J.M. Co-opting Moore’s Law: Therapeutics, Vaccines and Interfacially Active Particles Manufactured via PRINT®. J. Control. Release 2016, 240, 541–543. [CrossRef][PubMed] 12. Petersen, R.S.; Keller, S.S.; Boisen, A. Hot Punching of High-Aspect-Ratio 3D Polymeric Microstructures for Drug Delivery. Lab Chip 2015, 15, 2576–2579. [CrossRef][PubMed] 13. Abid, Z.; Strindberg, S.; Javed, M.M.; Mazzoni, C.; Vaut, L.; Nielsen, L.H.; Gundlach, C.; Petersen, R.S.; Müllertz, A.; Boisen, A.; et al. Biodegradable Microcontainers—Towards Real Life Applications of Microfabricated Systems for Oral Drug Delivery. Lab Chip 2019, 19, 2905–2914. [CrossRef][PubMed] 14. Abid, Z.; Mosgaard, M.D.; Manfroni, G.; Petersen, R.S.; Nielsen, L.H.; Müllertz, A.; Boisen, A.; Keller, S.S. Investigation of Mucoadhesion and Degradation of PCL and PLGA Microcontainers for Oral Drug Delivery. Polymers 2019, 11, 1828. [CrossRef] [PubMed] 15. Sachs, G.; Voegeli, H.E. Principles and Methods of Sheet-Metal Fabricating; Van Nostrand Reinhold: New York, NY, USA, 1966. 16. Lu, X.; Lozano, R.; Shah, P. In-Situ Dissolution Testing Using Different UV Fiber Optic Probes and Instruments. Dissolution Technol. 2003, 10, 6–16. [CrossRef] 17. Dash, T.K.; Konkimalla, V.B. Poly--Caprolactone Based Formulations for Drug Delivery and Tissue Engineering: A Review. J. Control. Release 2012, 158, 15–33. [CrossRef][PubMed] 18. Petersen, R.S.; Keller, S.S.; Hansen, O.; Boisen, A. Fabrication of Ni Stamp with High Aspect Ratio, Two-Leveled, Cylindrical Microstructures Using Dry Etching and Electroplating. J. Micromech. Microeng. 2015, 25, 055021. [CrossRef] 19. Fox, C.B.; Kim, J.; Le, L.V.; Nemeth, C.L.; Chirra, H.D.; Desai, T.A. Micro/Nanofabricated Platforms for Oral Drug Delivery. J. Control. Release 2015, 219, 431–444. [CrossRef][PubMed] 20. Nielsen, L.H.; Rades, T.; Müllertz, A. Stabilisation of Amorphous Furosemide Increases the Oral Drug Bioavailability in Rats. Int. J. Pharm. 2015, 490, 334–340. [CrossRef][PubMed] 21. Petersen, R.S.; Nielsen, L.H.; Rindzevicius, T.; Boisen, A.; Keller, S.S. Controlled Drug Release from Biodegradable Polymer Matrix Loaded in Microcontainers Using Hot Punching. Pharmaceutics 2020, 12, 1050. [CrossRef][PubMed] 22. Dey, N.; Majumdar, S.; Rao, M. Multiparticulate Drug Delivery Systems for Controlled Release. Trop. J. Pharm. Res. 2008, 7, 1067–1075. [CrossRef]