Lithography based methods to manufacture biomaterials at small scales

Besides being popular in microelectronics, lithography techniques have demonstrated a great potential use for drug delivery, tissue engineering, and diagnostic tools.. Introduction The u

Review Article

Lithography-based methods to manufacture biomaterials at small

scales

Khanh T.M Trana, Thanh D Nguyena,b,*

a Department of Mechanical Engineering, University of Connecticut, United States

b Department of Biomedical Engineering, University of Connecticut, United States

a r t i c l e i n f o

Article history:

Received 20 November 2016

Received in revised form

30 November 2016

Accepted 11 December 2016

Available online 21 December 2016

Keywords:

Lithography

Biomaterials

Intelligent therapeutics

Drug delivery

Tissue engineering

Biosensors

a b s t r a c t Along with the search for new therapeutic agents, advanced formulation and fabrication of drug carriers are required for better targeting, sensing, and responding to environmental stimuli as well as maxi-mizing treatment efficiency The emergence of intelligent therapeutics involves the use of functional biomaterials to mimic biological system for prolonged circulation and to work harmoniously with the body One of the main concerns lies in the feasibility of creating systems with well-defined architectures including size, shape, components, and functionality This review provides an overview regarding current challenges and potential of manufacturing and fabrication of biomaterials at small scales for various biomedical applications Accordingly, novel lithography-based fabrication approaches are introduced together with their remarkable applications Besides being popular in microelectronics, lithography techniques have demonstrated a great potential use for drug delivery, tissue engineering, and diagnostic tools

© 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

1 Introduction

The use of biomaterials is ubiquitous in biomedical applications

which include drug delivery systems, engineered tissues, and

biomedical devices Biomaterials are defined as synthetic or natural

materials which are in contact with biological environment in order

to treat any malfunctions of the body[1] Accordingly, biomaterials

must function synergistically with the body and possess sufficient

biocompatibility to avoid the risk of being recognized and

elimi-nated by the immune system Biocompatibility which is described

as“the ability of a material to perform with an appropriate host

response in a specific application” by Williams[2]is prerequisite

for any materials operating in human body Biomaterials are

tailored in such a way that they are able to resist the immune

response, blood clotting, or bacterial colonization Biomaterials

have been widely investigated and implanted in human body for

applications such as skeletal repair, organ replacement, and

improvement of senses, among many others with remarkable

success Artificial hip joint is one of the most popular implants

employing biomaterials[3] Hip joint prostheses are composed of titanium, stainless steel, special high strength alloys, ceramics, composites and ultra-high molecular weight polyethylene The replacement of worn-out hip joint helps restore patient ease of movement Likewise, heart valve prostheses also contribute to treat cardiac abnormalities [4] These implants are made of carbons, metals, elastomers, plastics, fabrics together with chemically pre-treated animal or human tissues to diminish immunologic activ-ities Other examples in this area include dental implants, cochlear replacement, contact lenses, etc.[5] These types of implants are expected to possess adequate protection from degradability in or-der to exhibit long term stability in the biological system On the other hand, biodegradability is essential for micro- and nano-systems to avoid invasive removal surgeries and possible toxicity innate to long-term implantation Furthermore, biodegradability can facilitate control over drug release profile

To this extent, the notion of intelligent therapeutics has evolved with the growth of biomaterials in response to the demand to manufacture improved functional systems Apart from their role as therapeutic carriers, these systems are created to be capable of advanced targeting or stimulating delivery, as well as detection/ diagnoses of diseases In the interest of fabricating intelligent therapeutics, these systems need to be responsive to a biological environment To do so, it is necessary for them to imitate the nature

* Corresponding author Department of Mechanical Engineering, University of

Connecticut, United States.

E-mail address: nguyentd@uconn.edu (T.D Nguyen).

Peer review under responsibility of Vietnam National University, Hanoi.

Contents lists available atScienceDirect Journal of Science: Advanced Materials and Devices

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d

http://dx.doi.org/10.1016/j.jsamd.2016.12.001

2468-2179/© 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license

Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

of their surrounding in terms of characteristics, sizes, and

struc-tures, which together present compelling challenges for scientists

to overcome So far, significant chemical progresses have

contrib-uted to the success of producing micro- or nano-sized bio-systems

that are suitable for certain kinds of diseases Current attempts have

mainly involved the employment of polymers and lipids to form

drug particles through chemical cross-linking, emulsion,

self-assembly, and dispersion alongside with chemical moiety modi

fi-cation for enhancing penetration, prolonging circulation, and

controlled delivery[6,7] However, particular difficulties remain in

synthesizing precise architectures with regard to size, shape, and

components as well as manipulating functionalized properties

[8,9] Recent works have explored physical micro- and

nano-fabrication methods in addition to conventional chemical ones,

offering superior techniques for drug delivery, tissue engineering,

biosensing, and disease-diagnostics

Lithography is a micro- and nano-fabrication technique that

enables formation of precise and complicated two-dimensional or

three-dimensional structures at extremely small scales The

tech-nique originated from a method of planographic printing on smooth

surfaces of a plate or stone This method was invented by the work of

a Bavarian author, Alois Senefelder, in 1976[10] The application of

lithography has advanced thefield of electronics by enabling the

mass production of semiconductors, electronic components and

integrated circuits[11] In the early twentieth century, there was a

limited use of photolithography for biomedicine despite the

devel-opment of advanced techniques in micro-patterning This was

mainly due to the high cost, complex operation and inaccessibility of

photolithography techniques to scientists[12] Later on however,

momentous growth of lithography has occurred due to increased

access to fabrication tools (clean-room facilities) in many research

institutes as well as lowering costs of production This further

became a driving force to expand lithography for studies in science

and technology, especially in biomedical areas [13] Early works

include the development of Bio-microelectromechanical systems

(Bio-MEMS), nanoelectromechanical systems (NEMS),

micro-fluidics, photonics, optics, and multifunctional devices Moreover,

the advantages of creating well-controlled morphology present

alternative opportunities to create intelligent therapeutics;

subse-quently, there is a significant interest in further exploration of these

approaches

This review aims to provide the current status of

chemically-functionalized biomaterials at micro- and nano-scales and further

describe advances in employing lithography-based micro- and

nano-fabrication methods, producing biomaterials for medical

applications

2 Functionalized biomaterials for biomedical applications

2.1 Functionalized biomaterials with certain physical and chemical

properties to cross biological barriers

In the attempt to administer therapeutic agents, the foremost

concern is their ability to bypass natural barriers The presence of

these barriers primarily serves as ultimate regulating entrances of

solutes and compounds; thereby, protecting the body against

in-vasion of foreign factors One of the most restrictive barriers in

human body to be mentioned is blood brain barrier which only

permits diffusional transport of small lipid soluble molecules with

molecular weight under 400 Da[14,15] The gastrointestinal barrier,

although viewed as a highly vascularized surface, presents selective

uptake activity by composing of enterocyte membranes, tight

junctions and specialized immunologic factors[16,17] Another type

of barrier is the stratum corneum (SC) situated on the upper layer of

skin SC is comprised mainly of multiple lamellar bilayer

corneocytes surrounded by an extracellular milieu of lipids; hence, it only prefers entrance of low molecular weight lipophilic com-pounds[18] These barriers together pose considerable challenges to thefield of drug delivery To deliver drug into desired organs, it is therefore desirable to deceive the barriers by creating drug carriers with special functions This requires the modulation of physical and chemical features of drug particles One of the initial efforts is to reduce particle size of drugs to the range of nano to a few microns There are two common approaches to fabricate nanoparticles in which the particles are built up from molecules (bottom up) or partitioned from larger ones (top down) Although small particles are proven to cross the barriers more effectively, nanoparticles with diameters smaller than 6 nm can be excreted by kidneys [19], whereas those larger than 200 nm might accumulate in the spleen and liver Nevertheless, the particle size is not the only concern in designing a drug delivery system In a review by Albanese and co-workers[20], they studied the correlation between the properties of nanomaterials (size, shape, chemical functionality, compositions, and surface charge) and theirs biomolecular signal, kinetics, distri-bution, and toxicity For instance, Yan Geng et al.[21]showed that rod-shape micelles could prolong circulation when compared to spherical ones Also, the surface charge of particles have effects on blood half-life Neutral nanoparticles possess the highest circulation time whereas other charges result in fast clearance, or interact with proteins (e.g immunoglobulin, lipoproteins) occurring in body fluids causing hemolysis, platelet aggregation, and coagulations

[22] Besides efforts to modify physical properties of therapeutic particles, functionalized particles can promote transport across different biological barriers[23] These include the employment of uptake-facilitating ligands such as apolipoprotein E (ApoE)[24]and transferrin[25]for drug delivery to brain or co-administration with P-glycoprotein inhibitors[26]for oral delivery or penetration en-hancers[27,28]for transdermal delivery

2.2 The use of biomaterials for controlled drug delivery Since each of therapeutic agents possesses unique properties in terms of water-solubility, crystallinity and chemical characteris-tics, they need to be encapsulated into appropriate carriers or biomaterials prior to manufacturing in order to meet individual compatibility and maximal drug-efficiencies Several techniques to formulate drug delivery systems are comprehensively developed, namely liposomes[29], micelles[30], emulsions[31,32], solid lipid nanoparticles [33], solid dispersion [34,35], drug-cyclodextrin complexation [36,37], and prodrugs [38,39] Due to the poor water-solubility of some therapeutic agents and the nature of biological barriers, the formulated systems usually possess certain degree of hydrophobicity Additionally, these formulations require extensive use of additives for stabilization and prevention of coalescence These additives are, however, associated with po-tential toxicity This concern demands the implementation of biomaterials with Food Drug Administration (FDA)’s approval and biodegradability The search for sources of natural and synthetic polymers suitable for biomedical applications has been widely carried out[40]

Designs of drug delivery systems have to be in accordance with different routes of administration and purposes of treatment A challenge for most biomaterials entering the body is the risk of being eliminated In a common approach, rapid clearance by phagocytic cells of mononuclear phagocyte system can be avoided

by attaching polyethylene glycol (PEG) to surface of the particles This process, alternatively so-called PEGylation, prevents opsoni-zation, thereby enhancing blood half-life of biomaterials[41] PEG

is approved by the FDA for usage in foods, cosmetics, and phar-maceuticals with little toxicity and is able to be further eliminated K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

in urine or feces[42,43] Pegylated liposomes that form hydrated

cover have shown positive impacts not only on extending

circula-tion time, increasing half-life, and decreasing plasma clearance by

protection from proteins and reticuloendothelial uptake but also

better drug encapsulation and leakage prevention, thereby

maxi-mizing treatment efficacies[44,45]

2.3 The use of biomaterials for tissue engineering

Biomaterials contribute to engineered tissues to replace parts of

the human body and harmoniously function with biological systems

[1] There exist several interests to engineer tissue properties and

performance by using biomaterials The interference of

tissue-engineered products with the extracellular fluids is significant

since it would trigger an inflammatory response Considering

po-tential long-term toxicity, biomaterials which have a degradability

rate matching with tissue regeneration rate are desirable In

accor-dance with dysfunctions of native organs, the implanted tissues

would preferably start to operate at time of treatment to replace

impaired ones Alternatively, they may convert into expected form

upon implantation [46] Thus, the composition, architecture, 3D

environment of the scaffold, and biocompatibility of materials are

challenging factors that strongly impact formulation of implanted

tissues[47] Biomaterials should be processable to complex shapes

with porosity and sufficient mechanical strength[48] Moreover,

consistency and uniformity are requirements to manufacture

tissue-engineered products Also, tissue and cell preservation from

func-tion loss during long-term storage needs to be improved[49] Efforts

have also been made to mimic the extracellular matrix for tricking

the body immune response A number of natural materials have

been employed to this end including alginate[50], chitosan[51],

hyaluronic acid[52], etc Another attempt is to incorporate signal

peptides (RGD) into materials[53]

3 Chemical modulation of biomaterials

Numerous efforts have been made to study and obtain desired

properties of biomaterials for different medical applications

Chemical methods have been tremendously used to successfully

modulate biomaterials by adding or combining functional chemical

groups, which can response to specific stimuli and environments

As such, we briefly cover some important chemical modulations

before describing in more details lithography-based technology as a

powerful supplemental approach to manufacture biomaterials

3.1 Responsive-biomaterials

Considerable amounts of agents used for pharmacotherapy may

exhibit side-effects besides advantageous activity when delivered to

wrong targets or healthy tissues This phenomenon is usually

pre-sent in cancer therapy in which cytotoxic compounds can kill

normal cells in addition to cancer cells Efforts have been made

to-ward chemical modifications of biomaterial carriers' structures and

surfaces for specific delivery or drug targeting[54] The implications

of stimuli-responsive biomaterials propose an opportunity to

fabricate active drug carriers that can release therapeutic agents

through particular triggers including biological stimuli such as pH,

temperature, redox microenvironment or artificial stimuli such as

light, magnetic, etc In a review by Ganta et al.[54], various

stimuli-responsive nano-systems are comprehensively discussed

Accord-ing to Caldorera-Moore et al.[55], responsive biomaterials can be

categorized into two groups Thefirst group refers to passive carriers

which respond to external conditions (pH, thermodynamic, ionic

strength, magnetic, and electrical) by physicochemical interactions

On the other hand, functionalization of materials can be performed

to form ligand receptors which interact and act in response to bio-markers or bioanalytes present in a medium or diseased tissue 3.2 Polymer-based biomaterials

Many polymer-based biomaterials have been widely studied Among them, crosslinking hydrophilic polymers or hydrogels appear

as one of the most potentially effective systems for medical appli-cations since they permit the incorporation of functional groups directly into theirs networks Additionally, the high water affinity and swelling property of hydrogels are believed to importantly contribute to their interesting behaviors towards environmental stimuli The working mechanism of such responsiveness relies on the side chain groups, branches, and crosslinking structures of polymeric materials The pH-sensitive hydrogels consisting of pendant acidic and basic groups (e.g carboxylic, sulfonic acids, ammonium salts) can simultaneously accept or release protons corresponding to medium pH through movement of solutions into the networks[56]as presented inFig 1 Some of the most studied polymers for this area to be named are poly(acrylic acid) (PAA)[57], poly(N,N9-diethylaminoethyl meth acrylate) (PDEAEM)[58], pol-y(methacrylic acid) (PMA)[59], carrageenan, alginate[60]etc The pH-sensitive hydrogels have been applied to control drug release rate in a gentle manner for specific sites such as gastro-intestinal

[61e63], transdermal[64e66] A recent review by Karimi and co-workers[67]has thoroughly addressed several stimulus-responsive nano-carriers for controlled drug release Besides pH sensitivity, other environmentally sensitive systems have been investigated

[68] Several polymers possess reversible phase transitions upon temperature variations owning to the presence of hydrophobic groups namely methyl, ethyl, and propyl[69] Different polymers have been widely studied for these works are poly (N-iso-propylacrylamide)[70], poly (N-vinylcaprolactam)[71] Efforts have been made to create multi-functional systems for better drug transporting and targeting[72e74] Furthermore, hydrogels have presented great applications for tissue engineering scaffolds which are discussed in several papers[7,75,76]due to their high density and structural support while preserving in vivo environment Another polymer-based system is emulsified microparticles, so-called microemulsions Microemulsions are isotropic, thermodynamically stable systems of oil, water, and surfactant, frequently in combina-tion with a co-surfactant[77] The performance of microemulsion as drug delivery systems is remarkable Their low surface tension and small droplet size enhance the absorption and permeation rate through membranes Solid dispersion has also been studied as an effective strategy for drug delivery systems The process creates active ingredients dispersed in an inert carrier matrix [78] The enhanced dissolution rate of drug by solid dispersion may contribute

to the increased drug solubility due to the reduction of the dispersed particle size, conversion from crystalline to amorphous state, and drug wetting improvement The two most common methods used to produce solid dispersion are melting and solvent evaporation Several polymers that have been tested as carriers for solid disper-sion systems, including PEG, polyvinyl pyrrolidone (PVP), cellulose derivatives, polyacrylates, and polymethacrylates, among many others[79].Table 1lists different lithography-based methods which could be used to manufacture such polymer-based biomaterials 3.3 Limitations of current chemical methods and the need of lithography-based technology

Despite tremendous advantages and achievements of chemical methods, the clinical utilizations of chemically-engineered nano-and micro-carriers have been limited by the difficulties associating with uniformity and consistency in terms of controlling specific K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

size, shape, chemical components, and functionality[80,81] For

instance, self-assembling structures such as liposomes, micelles,

emulsions exhibit a dynamic instability, thereby presenting

chal-lenges in manipulation of exact size, shape, and drug encapsulation

or dosing[82,83]

The emergence of lithography-based techniques to engineer

micro- and nanocarriers and to produce better controlled system

properties has offered a promising alternative in the field of

biomedicine One of recent papers in our group has thoroughly

addressed up-to-date applications of advanced 3D technologies to

create well-structured micro- and nano-carriers for controlled drug

delivery system[84]

4 Lithography technology

4.1 Photolithography

Photolithography or optical lithography is defined as a process

using light to transfer patterns from a photomask to a photoresist

(light-sensitive chemical) on a substrate and then selectively

remove unused parts out of the substrate Photolithography

tech-nique is based on a top down approach Different processing

pro-tocols and materials are required for different implementations of

photolithography; however, they largely follow a basic common

procedure as presented inFig 2 To prepare for photoresist coating,

a substrate like silicon wafer must be removed of any contaminants

including solvents stains (methyl, alcohol, acetone, etc.), dust from atmosphere, operators, and equipment, microorganism, aerosol particles, etc on the surface[85,86] The process requires operation under cleanroom facilities enclosed in a strictly environmentally controlled space in terms of airborne particulates, temperature, air pressure, humidity, vibration, and lighting[87] For certain cases especially in biomedical applications, the silicon wafer basically serves as a solid support on which additional layers of materials are deposited due to its ideal characteristics namely rigid,flat, low cost, and smooth[13] The wafer is coated with a thin layer of photo-reactive materials that generally are monomers, oligomers, or polymers For patterning biomaterials like proteins and cells, near-infrared (NIR) light is more preferable than UV since it is less photo-damaging and deeper penetration[88] To this extent, depending

on the nature of photoresists, there are different ranges of radiation which can be used such as electron beams, ion-beam, and X-ray The fundamental principle of photolithography lies in the chemical alterations of the resist upon light exposure[89] By shining UV light through a photomask which consists of non-transparent patterns printed on a transparent plate, the patterns are trans-ferred onto the photoresist In the next developing step, the remaining parts of the photoresist after exposure relied upon whether positive or negative photoresist is employed which sub-sequently dissolve exposed and unexposed regions respectively Photolithography has established a fundamental foundation for further development of other advanced methods (seeFig 3)

Fig 1 Diffusion of drug in response to pH variations [166]

Table 1

Lithography-based technology in forming polymer-based biomaterials.

Lithography-based

technology

Polymer-based biomaterials

Photolithography Proteins, cells,

extracellular matrices

Photon upconversion lithography (PUCL)

Providing high resolution in noncontact manner which prevents contamination.

Less photo-damage by the use of near infrared light and deeper penetration into tissues.

Large scale pattering.

[152]

Soft lithography Emulsified polymeric

systems

Microfluidic devices Systems with better control over size, structure, and

composition.

Scalability, low cost, reproducibility, and high throughput

[146]

Photolithography Solid dispersion

polymeric systems

PVP solution was dispensed into lithographically patterned microcontainers.

Ketoprofen was impregnated in polymer matrix by using supercritical carbon dioxide e loading medium.

Higher efficiency when compared to conventional dispersing methods.

Higher precision of drug dosing is obtained together with better dissolution results.

[171]

K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

4.2 Advanced lithography-based methods

4.2.1 Soft lithography

Despite the fact that photolithography is prevalent in both

mi-croelectronics and biomedicalfields, there exists certain limitations

restricting it from being suitable for all applications For example,

the process of photolithography requires expensive facilities

(cleanroom, photomasks fabrication, projecting systems) The

conventional lithography techniques, although being

well-established in semiconductors industry, encounter noticeable

im-pediments owning to the rigorous processes Several

manufacturing steps such as cleaning, baking, exposing, which

require the presence of high-temperature, ion-etching, solvents,

often result in degradation of biomaterials [90] Additionally,

photolithography has neither control over surface chemistry nor

implementations on curved/non-planar surface[91] Based on the

conventional method of lithography, scientists have developed an

alternative set of micro-fabrication working on “soft-matter”

(organic materials, polymers, complex biochemical) and the pattern-transfer by molding using elastomeric biomaterials named

as soft lithography [92,93] The method of soft lithography is fundamentally based on printing, molding, and embossing and has been extensively described in many literatures [12,91,94] Soft lithography involves techniques of using elastomeric stamps, molds, and photomasks to fabricate or replicate structures [94] Some of the most well-studied patterning techniques are micro-contact printing (mCP)[95], replica molding (REM)[96], micro- and nano-transfer molding [97,98], solvent-assisted micromolding (SAMIM)[99], phase-shifting edge lithography[100], decal transfer lithography[101], and nanoskiving[102]

4.2.2 Nanoimprint lithography Nanoimprint lithography is defined as the process of pressure-induced transferring of patterns from a rigid mold to a thermo-plastic polymerfilm heated above its glass transition temperature This method alternatively refers as hot embossing Recent review

Fig 2 Schematic of photolithography [167] (a) The wafer is cleaned to remove any unwanted contaminants (b) The photoresist is spin coated onto the wafer (c) The photomask is placed above the photoresist UV light is exposed through the mask (d) The unexposed part is removed by solvents leaving the desired patterns.

Fig 3 Schematic process of (a) polymer mold fabrication from a master and (b) UV nanoimprint with the polymer mold Reprinted from reference [168] with permission from Elsevier.

K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

by Traub et al [103] has extensively discussed the principle of

nanoimprint lithography and its wide applications in different

fields Essentially, the nanostructured mold is duplicated in a thin

resist film casted on a substrate by applying adequate pressure

[104] This stage involves heating the resist above its glass

transi-tion temperature where it becomes viscous and deformable into

shape The mold is then removed together with residual resist in

the compressed parts Since the process is associated with heat and

pressure, it is important to select appropriate resist materials which

have relatively small thermal expansion and pressure shrinkage

coefficients Bender et al.[105]first introduced a modified

nano-imprint lithography operating at room temperature and low

pres-sure, which is based on UV-irradiation and photopolymerization

(Fig 1) Similarly, the polymer UV-transparent mold (quartz or

silica) is pressed onto the substrate coated with UV-curable resist

Following exposure, the precursor liquid crosslinks and constructs

stable patterns Ever since introduction, nanoimprint has made a

significant progress due to its simplicity, efficiency, high

throughput, and low production cost Moreover, research

pertain-ing to thefield includes enhancing resolutions of the patterned

nanostructures[106] To this extent, Koo et al.[107]investigated

the effects of mold materials on the fabrication process Their works

showed that toluene diluted poly(dimethylsiloxane) (PDMS) was

capable of homogeneously defining 50 nm resolution patterns on 4

in wafer with single imprint Other concerns and applications

regarding nanoimprint techniques to fabricate biomaterials have

been addressed in several literatures[108,109]

4.2.3 Nano-molding PRINT particles

The method of Particle Replication In Nonwetting Templates

(PRINT) wasfirst developed by DeSimone et al in 2004 Ever since,

it has drawn significant attention from scientists due to its ability to

generate monodisperse micro- and nano-carriers with

simulta-neous control over particle size, shape, composition, and

func-tionalities by employing the advances of soft lithographic molding

technology The initial effort in the PRINT process is to formulate

particle composition liquid which consists of essential

chemo-therapeutics or functionalized features Different particles ranging

from 80 nm to 20mm, composed of poly(D-lactic acid) (PDLA) and

derivatives[81], PEG hydrogels[110,111], and proteins[112]have

been successfully manufactured Secondly, the solution is then

placed onto a prepared mold and allowed for solvent evaporation

or polymerization The stage involves the process to pattern replica

molds with desired size and shape to emboss liquid precursor

compounds In this regard, the silicon master templates werefirstly

constructed by patterning silicon wafer coated with poly(methyl

methacrylate) (PMMA) resist using e-beam lithography

Subse-quently, photocurable perfluoropolyether (PFPE) was pooled onto

the templates and chemically cured to form elastomeric PFPE

molds[113] PFPE appears to be one of the most suitable materials

because of its nonwetting and nonswellingfluorinated surfaces to

both organic and nonorganic solvents[114] This enables the

cre-ation of isolated, harvestable“scum-free” particles without harsh

processes With this in mind, thefinal step following mold filling is

to harvest the particles One physical method uses the sharp end of

a glass side to remove the particles; however, numbers of

disad-vantages arise that are damaging the mold surface, aerosolizing dry

particles, and inability to scale the process Hence, a gentler process

was investigated by laminating an adhesive release layer such as

PVP or cyanoacrylate on aflexible or rigid backing PET or glass

slides on the open side of the mold After that, the mold is peeled

away, leaving the array of free-standing particles Dissolution of the

adhesivefilm results in free-flowing particles in solutions [115]

Additional purification steps are performed accordingly such as

dialysis, centrifugation,filtration, and magnetic purification in or-der to eliminate residual or debris chemicals[113,116]

5 Applications of advanced lithography-based methods for biomedicine

5.1 Drug delivery systems 5.1.1 PRINT particles PRINT has offered an advanced lithography-based method to operate with a wide range of organic materials containing biolog-ical elements such as oligonucleotides, proteins, pharmaceutbiolog-icals, and synthetic viruses In a studied by Gratton et al.[113], cellular internalization, cytotoxicity of monodisperse 1 mm cylindrical PRINT particles and the effect of surface charge on endocytosis were investigated using confocal microscopy,flow cytometry, and transmission election microscopy Fig 4 describes the result of PRINT fabrication process The particles were readily distributed into tested cells: HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 with little cytotoxicity obtained Also, higher endocytosis rate was observed in particles with positive zeta potential as compared to negative ones On-going research in thefield focuses on formu-lating stimuli response targeted nanoparticles[117,118] All in all, PRINT e a highly versatile method - has become uniquely suitable for large scale manufacturing monodisperse organic and non-organic nanoparticles with precise size, shape, composition, sur-face properties dedicating to applications in nanomedicine 5.1.2 Nanoimprint applications

Nanoimprint is suitable for various polymeric materials such as biomolecules[119,120], block copolymers [121,122] with feature sizes down to 5 nm and high aspect ratios In a remarkable study of Glangchai et al.[8], by employing nanoimprint techniques, they were able to synthesize enzyme-triggered release nanoparticles of antibodies (Streptavidin-CY5) and nucleic acids (plasmid DNA) with well-defined sizes and geometries (square, triangular, pentagonal) The responsive and biocompatible properties were obtained through combination of PEG diacrylates (PEGDA) or dimethacrylates (PEGDMA) polymers and an acrylated, enzymati-cally degradable peptide Gly-Phe-Gly-Lys-diacrylated (GFLGK-DA) Additional biocompatible photoinitiator (2-hydroxyl-1-[4-(hy-droxyl)phenyl]-2-methyl-1-propanone) was added to trigger pho-topolymerization when performing UV exposure Nanoimprinting was conducted using the Step and Flash Imprint lithography (S-FIL) method (Fig 5) After imprinting, the nanoparticles were isolated

by reactive ion etching (RIE), removing residual areas between particles And the nanoparticles were collected into poly(vinyl alcohol) (PVA) solutions Elucidations and drug release studies demonstrated the efficiency of nanoimprint to fabricate long-circulating nanocarriers as small as 10 nm with controlled size and shape which responded to environmental stimuli Also, the method proved its mildness for biological agents without the use of high temperature, high shear, extended UV exposure, and organic solvents The presence of available groups to attach specific ligands

on the chemical structures of formulated materials offers great opportunities for targeted drug delivery and imaging[123] 5.1.3 Microneedles

Transdermal delivery emerges as a potential route of adminis-tration since it offers a direct drug application to an affected site, steadier drug concentration in plasma, and limits systemic expo-sure by the absence of hepaticfirst pass metabolism[124] Yet, the presence of stratum corneum (SC) as skin barrier functions as the most significant regulator for entry into the body SC prevents entrance of therapeutic agents except for that of lipophilic and low K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

weight molecules Efforts have been made to formulate in situ

topical applications, particularly patches [125], gels, and creams

[126] Some of the well-studied systems in thisfield are available in

the market[127] However, these conventional systems possess

certain disadvantages namely high viscosity, lack of flexibility,

visibility, and inadequate retention on skin which then requires

repetitive dosing and poor patient compliance For the past decade,

microneedles have provided a straightforward method to directly

administrate therapeutic agents bypassing the SC in a

minimally-invasive manner [128] The system only needs to pierce pass

10e15mm nerve-free layer for drug to diffuse through the highly

permeable viable epidermis to capillaries Unlike regular

hypo-dermic needles, microneedles create micro-dimensional painless

pathways to transport small drug particles, macromolecules,

proteins, andfluid with a high localization, correct-targeting, and controlled release The development of microneedles has been accompanied by the revolution of lithography methods in biomedical research

The first attempt to produce microneedles based on photoli-thography techniques was reported by Henry et al in 1998[129] In this process, 〈100〉-oriented, prime grade, 450e550 mm thick, 10e15U-cm silicon wafers supplied by Nova Electronic Materials Inc (Richardson, TX) were initially cleaned in a mixture of deion-ized water, hydrogen peroxide, and ammonium hydroxide at approximately 80 C for 15 min, and followed by dehydration baking at 150 C for 10 min Chromium was deposited onto the wafers and patterned into 20 20 arrays of 80mm diameter dots with 150 mm center-to-center spacing by UV exposure of the

Fig 4 Results of the PRINT process Top row, left to right: a) SEM of an etched silicon wafer master template of 3mm posts having a height of 1.7mm; b) cured PFPE mold of the master template shown in A; c) PFPE mold containing PEG particles prior to harvesting; d) harvested and dispersed PEG PRINT particles Bottom row, left to right: e) SEM of an etched silicon wafer patterned with approximately 400 billion posts that are 100 nm in diameter and 400 nm tall; f) a cured PFPE mold of the silicon master template shown in E; g)

100 nm PEG particles made using PRINT and transferred to a medical adhesive layer for surface functionalization and subsequent harvesting Reprinted from reference [169] with permission from Elsevier.

Fig 5 Step and Flash Imprint lithography (S-FIL) method: 1 PVA release layer and PEGDA is applied to BARC coated silicon surface 2 The quartz template is pressed onto PEGDA and exposed to UV light 3 The template is removed to reveal particles with thin residual layer 4 Brief oxygen plasma etch is performed to remove residual layer 5 Particles are harvested directly in water or buffer by one-step dissolution of the PVA layer Reprinted from reference [8] with permission from Elsevier.

K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

photoresists coated on a chromium layer Through immersing the

wafers to a liquid developer, the exposed photoresists were

removed Then the chromium exposed during previous

photoli-thography was etched, revealing dot arrays of chromium on the

silicon wafers which were further used as masks for microneedle

fabrication Solid microneedles were manufactured by deep

reac-tive ion etching (RIE) silicon substrates with 20 standard cm3/min

(sccm) SF6and 15 sccm O2 at a pressure of 20 Pa and power of

150 W for roughly 250 min[130] The parts covered with chromium

formed the microneedles The etching was continued until the

masks completely under-etched and fell off, forming arrays of sharp

silicon spikes From this early study, microneedles demonstrated

their potential in painlessly piercing into skin without breaking or

disrupting skin nature Additionally, in vitro results indicated

enhanced calcein permeability by 25000-fold and prolonged

release for 5 h Microneedles have opened a new perspective for

transdermal delivery

Despite the fact that silicon possesses comprehensive pro-cessing experience, it is considered to be fragile, arguably biocompatible, and comparably expensive[131] Later works have employed metals [132], glass [133], ceramics [134], and biode-gradable polymers [135] to fabricate microneedles in different shapes and sizes for specific applications Solid microneedles were later manufactured to possess beveled-tip, chisel-tip, and tapered-cone needles as presented in the work of Park et al.[131] The fabrications are fundamentally based on conventional photolith-ographic process, etching, laser cutting, metal electroplating, electropolishing, and micromolding [136e139] A review by McAllister and co-workers [140] has extensively described different approaches using metals and polymers for transporting macromolecules and nanoparticles Generally, microneedles are classified into solid microneedles including drug-coated micro-needles, dissolving microneedles and hollow microneedles (Fig 6) Properties of each kind are discussed in Table 2 For instance,

Fig 6 (a) Solid microneedles, (b) Coated microneedles, (c) Dissolving microneedles, (d) Hollow microneedles Reprinted from reference [170] with permission from Elsevier.

Table 2

Comparison of different microneedles fabricating approaches [170]

Types of microneedles Drug loaded Release mechanism Limitations Applications

Solid

microneedles

Coated

microneedles

[172]

Dipping or spraying drug-formulated solution onto solid microneedles Additional use of surfactants to facilitate wetting process and stabilizing agents, which protect drug from drying and storage.

Drug coated on microneedles dissolves into tissues upon insertion and contact with biofluids.

Limit to drugs with small doses.

Coating solutions should possess water-solubility, good mechanical resistance, and pharmaceutical acceptance.

Small molecules: vitamin B, fluorescein [173]

Macromolecules: insulin

[174] , verapamil hydrochloride and amlodipine besylate [175] , epigallocatechin-3-gallate

[176] , hormone [177] , bovine serum albumin

[178] , desmopressin [179]

Vaccines [180] : hepatitis B surface antigen [181] , influenza virus [182] , human papillomavirus

[183] , measles vaccination

[184] , inactivated chikungunya virus [185] , hepatitis C DNA vaccine

[186] , herpes simplex virus

[187]

Dissolving

microneedles

Therapeutics are encapsulated in formulated polymers which are able to solidify or polymerize during micromolding.

The polymer microneedles completely dissolve or degrade

in the skin as responding to stimuli (temperature, pH, solvents), and leaving no bio-wastes after administrations.

Some formulations require long remained time on skin to sufficiently dissolve Materials and fabrication methods should be carefully tailored for specific therapeutic agents.

Sulforhodamine [188] , insulin [189] , erythropoietin [190] , human growth hormone

[191] , Hepatis B vaccination [192]

Hollow

microneedles

Therapeutics especially in liquid formulations are entrapped in the reservoir and injected through the hollow space of microneedles.

The flow of liquid through microneedles is generated using a syringe of actuators that are controlled by CO2 gas pressure, a spring, a piezo-electric

micropump, a piezoelectric linear servo motor, a syringe pump and a micro-gear pump

Fluid flow rate in certain cases might depends on insertion depth, pressure, needle tip shape, and spreading factor [195] Hollow microneedles require more advanced fabrication techniques than previously mentioned ones.

Insulin [196] , doxorubicin

[197] , phenylephrine [198] , vaccine [199] , inactivated polio vaccine [200]

K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

hollow microneedles have been developed as means of delivering

insulin and vaccines by infusion [132] In fact, hollow

micro-needles have adapted techniques in MEMS fabrication Similarly,

photoresist was deposited and patterned onto silicon wafers

Straight-walled holes were yielded by utilizing Bosch modified

inductive coupled plasma reactive ion etching (ICP-RIE) [141]

Another interesting work by Kim et al.[142]designed a responsive

system to separate microneedles into skin upon contacting the

biofluid, which was triggered by swelling of hydrogels The

poly-Nisopropylacrylamide (PNIPAAm) hydrogels microparticles

pre-pared by emulsification method were filling into the cavities of the

mold prior to microneedle construction by micromolding

poly-lactic-coglycolic acid (PLGA) The systems were studied in vivo

by inserting microneedles to porcine cadaver skin Sustained drug

release evaluation was performed in vitro through Franz cell

model The drug release mechanism was due to the dissimilar

degrees of swelling among hydrogels and needle matrix polymer

causing cracking of microneedles In vivo study on mouse skin has

also drawn analogousfindings that the formulated microneedles

successfully released drug into the skin Collectively, fabrication of

microneedles for transdermal drug delivery using

lithography-based method has been on the edge of development due to the

ability of microneedles to effectively drive drugs into skin in a

controlled and targeted manner together with high patient

compliance and ease of large scale production

5.1.4 Microfluidic devices to fabricate drug particles Microfluidic devices offer powerful tools to fabricate mono-disperse microparticles in a high throughput manner[143] The most common materials used to manufacture microfluidic devices are (PDMS) Employed in the photolithography fabricating process, PDMS enables formation of small scale and complex channels in the devices (Fig 7) Briefly, the SU-8 photoresist is coated in silicon wafer and patterned The resist structures are further used as negative mold masters to pattern PDMS The PDMS is poured over the mold master and cured for 1 h at 70C After being peeled off, the PDMS mold is attached to a glass slide for further serving as microfluidic devices [144] In a study by Xu et al [145], mono-disperse biodegradable drug-loaded microparticles were success-fully fabricated by microfluidic flow-focusing generators and rapid solvent evaporation from resulting droplets The particle size could

be modulated through controlling theflow conditions within the devices Drug release studies had shown that particles prepared by this method exhibited critical reduction in burst release effect and slower release rate in comparison with conventional emulsion methods; hence, presenting potential for prolonging drug release

Fig 7 Basic microfluidic device fabricating process Reprinted with permission from

reference [144] Copyright 2002 American Chemical Society. Fig 8 Schematics of bilayer embossing process The inset shows the top-view of the

K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14

Other drug delivery system formulated using microfluidic devices

are emulsion[146], microgels[147], chitosan-based nanoparticles

[148], polymeric microcapsules[149], and pH responsive polymer/

porous silicon composites system[150] Riahi et al.[151]discussed

current expert opinion in microfluidics for advanced drug delivery

All in all, this effective, simple, and inexpensive fabricated

micro-device is predicted to continue toflourish in biomedical research

5.2 Tissue engineering

Microfabrication techniques have been used to replicate

struc-tures with well-controlled microenvironments of individuals,

in-teractions of multiple cell clusters, and with sizes ranging from 0.1

to 10 mm Thus, microfabrication techniques are viewed as a

potentially effective approach to promote highly-organized

scaf-folds in tissue engineering Photolithography has been used to

pattern biomaterials such as proteins, cells, and extracellular

matrices[152] Soft lithography implements master molds such as

PDMS elastomers to product PLGA scaffolds[153e155] One study

by Yang et al.[156]developed a biologically benign CO2assisted 3D

scaffolds using micromolding Photolithography wasfirst used to

construct desirable patterns of SU-8100 on a substrate following by

dispersion of PDMS resin The inverse PDMS mold was peeled off

after 2 h curing at 65C In the next step, bilayer PLGA skeletons

were precisely patterned by being melt at 220 C and then

embossed with PDMS mold at 0.1e3 MPa as illustrated inFig 8 The

bonded scaffolds were created by pressured saturation with CO2at

0.69 MPa and low temperature for 1 h Cell culture study indicated

the cytocompatibility of scaffolds This work contributed a

power-ful, solvent-free, and low cost method to engineer well-defined

structure scaffolds However, possessing a common problem in tissue engineering, micro-fabricated tissue scaffolds have a limited control and ability to create effective microvascular systems within the scaffold structure[157]

The merger of microfabrication and hydrogels have indeed proposed great feasibility to overcome current limitations and open new functional applications in tissue engineering[55] A review by Khademhosseinia and Langer [158] has broadly discussed per-spectives of various hydrogels synthetic approaches especially microfabrication as well as its applications in tissue engineering For instance, shape-controlled cell-laden microgels fabricated by micromolding photocrosslinkable hydrogels are able to be seeded with diverse cell types and assembled to form 3D structures in highly-governed structures and cell interactions[159] Addition-ally, the microengineered hydrogels are of great benefits for their surface modifications Through covalently immobilized cell integ-rin ligand (ephinteg-rin-A1 and Arg-Gly-Asp-Ser), a vascular develop-ment factor, on PEG hydrogels surface using photopolymerization, the adhesion of endothelial cells is improved together with better control over angiogenic functions[160] A remarkable research of Yeh et al [161] also used a micromolding technique to entrap mammalian cells in 3D microscale photocrosslinked harvestable hydrogels of controlled size and shapes

5.3 Biosensors Photolithography has been employed to micropattern hydrogels for biological sensors due to the ability of hydrogels to capture a wide range of biological sensitive factors The 3D structures of microgels provide greater density of receptor molecules, hence improving sensing capacity when compared to 2D systems[162] For example, Bashir et al [163] patterned an environmentally-responsive antibody-laden hydrogel onto a MEMS microcanti-lever (Fig 9) When absorbing targeted proteins, this sensitive system swelled and deflected the MEMS cantilever The degree of

deflection was then calculated by refractive optics Investigations

on pH and thermal sensitive hydrogels were carried out and resulted in similarfindings[164] In another study, PEG hydrogels were developed as biotin-streptavidin biosensors by combining methods of surface graft polymerization and photolithography

[165] Modifications of protein-repellent PEG hydrogels surface were made by grafting poly(acrylic acid) (PAA) as monomers and

Fig 9 Cross-sectional schematic of the cantilever/polymer structure with the various

dimensions Reprinted from reference [163] with the permission of AIP Publishing.

Fig 10 Micropatterning of PAA on the PEG hydrogel surface Optical image of 100mm diameter circles of PAA on PEG hydrogel surface Reprinted from reference [165] with

K.T.M Tran, T.D Nguyen / Journal of Science: Advanced Materials and Devices 2 (2017) 1e14