Fabrication of Dissolving Polymer Microneedles for Controlled Drug Encapsulation and Delivery: Bubble and Pedestal Microneedle Designs

Dissolving microneedle patches offer promise as a simple, minimally invasive method of drug and vaccine delivery to the skin that avoids the need for hypodermic needles. However, it can be difficult to control the amount and localization of drug within microneedles. In this study, we developed novel microneedle designs to improve control of drug encapsulation and delivery using dissolving microneedles by (i) localizing drug in the microneedle tip, (ii) increasing the amount of drug loaded in microneedles while minimizing wastage, and (iii) inserting microneedles more fully into the skin. Localization of our model drug, sulforhodamine B in the microneedle tip by either casting a highly concentrated polymer solution as the needle matrix or incorporating an air bubble at the base of the microneedle achieved approximately 80% delivery within 10 min compared to 20% delivery achieved by the microneedles encapsulating nonlocalized drug. As another approach, a pedestal was introduced to elevate each microneedle for more complete insertion into the skin and to increase its drug loading capacity by threefold from 0.018 to 0.053 μL per needle. Altogether, these novel microneedle designs provide a new set of tools to fabricate dissolving polymer microneedles with improved control over drug encapsulation, loading, and delivery.     

日夜两用生长激素微针贴片的制备及体外评价

目的 制备日夜两用生长激素微针贴片，模拟人体生理状态下内源性生长激素分泌的昼夜差异，实现生长激素给药时间和用量的优化，同时有效减轻皮下注射给药疼痛感，提高患者使用依从性。方法 采用铸模法制备微针贴片，通过光学显微镜和扫描电子显微镜观察微针表面形貌。经体外释放试验确定含生长激素微针制备的最佳工艺条件，包括优化紫外交联时间和交联剂含量。通过微针力学强度测试和体外透皮试验验证微针贴片有效穿透皮肤的可行性，通过圆二色光谱测定药物的稳定性。通过调试负载不同剂量生长激素制备日用和夜用微针贴片。结果 在显微镜下观察到微针排列整齐，针体完整、尖锐，微针在药物释放前后形貌无明显差异。工艺优化结果表明，当紫外交联时间为7 min，交联剂用量为1.5%时，微针贴片可以有效穿透离体大鼠皮肤，同时实现了生长激素在12 h内稳定释放，且微针释放出的蛋白药物构象无明显变化。通过在针体中负载不同剂量生长激素，制备了日用和夜用生长激素微针贴片。结论 本研究制备的微针能够顺应在生理状态下生长激素分泌的日夜差异，实现了适宜时间释放适量生长激素的目标，未来可进一步优化微针药物负载量以满足不同患者的实际使用需求，以期实现个体化治疗。     

Challenges and strategies in developing microneedle patches for transdermal delivery of protein and peptide therapeutics

The birth of microneedles, an array of needles sufficiently long to penetrate epidermis but small enough to do not cause skin injury and pain feeling, has offered a highly promising solution for non-invasive delivery of protein and peptide drugs, a long-cherished desire over eighty years. However, the attempts to develop clinically feasible microneedle transdermal delivery methods encountered series of difficulties, for which a decade research efforts have yet to result in a single product. Microneedles may be incorporated into devices as skin pre-treatment tools, skin microinjectors as well as transdermal patches by their functions in drug delivery. They may also be categorized to insoluble solid microneedles, hollow microneedles, soluble/degradable solid microneedles and phase-transition microneedles by their structure and forming materials. This review article is aimed to update the progress and discuss the technical challenges raised in developing protein/peptide loaded microneedle patches.     

Design, Optimization and Characterisation of Polymeric Microneedle Arrays Prepared by a Novel Laser-Based Micromoulding Technique

Purpose

Design and evaluation of a novel laser-based method for micromoulding of microneedle arrays from polymeric materials under ambient conditions. The aim of this study was to optimise polymeric composition and assess the performance of microneedle devices that possess different geometries.

Methods

A range of microneedle geometries was engineered into silicone micromoulds, and their physicochemical features were subsequently characterised.

Results

Microneedles micromoulded from 20% w/w aqueous blends of the mucoadhesive copolymer Gantrez® AN-139 were surprisingly found to possess superior physical strength than those produced from commonly used pharma polymers. Gantrez® AN-139 microneedles, 600 ??m and 900 ??m in height, penetrated neonatal porcine skin with low application forces (>0.03 N per microneedle). When theophylline was loaded into 600 ??m microneedles, 83% of the incorporated drug was delivered across neonatal porcine skin over 24 h. Optical coherence tomography (OCT) showed that drug-free 600 ??m Gantrez® AN-139 microneedles punctured the stratum corneum barrier of human skin in vivo and extended approximately 460 µm into the skin. However, the entirety of the microneedle lengths was not inserted.

Conclusion

In this study, we have shown that a novel laser engineering method can be used in micromoulding of polymeric microneedle arrays. We are currently carrying out an extensive OCT-informed study investigating the influence of microneedle array geometry on skin penetration depth, with a view to enhanced transdermal drug delivery from optimised laser-engineered Gantrez® AN-139 microneedles.     

Polymer Microneedles for Controlled-Release Drug Delivery

Purpose As an alternative to hypodermic injection or implantation of controlled-release systems, this study designed and evaluated biodegradable polymer microneedles that encapsulate drug for controlled release in skin and are suitable for self-administration by patients. Methods Arrays of microneedles were fabricated out of poly-lactide-co-glycolide using a mold-based technique to encapsulate model drugs—calcein and bovine serum albumin (BSA)—either as a single encapsulation within the needle matrix or as a double encapsulation, by first encapsulating the drug within carboxymethylcellulose or poly-l-lactide microparticles and then encapsulating drug-loaded microparticles within needles. Results By measuring failure force over a range of conditions, poly-lactide-co-glycolide microneedles were shown to exhibit sufficient mechanical strength to insert into human skin. Microneedles were also shown to encapsulate drug at mass fractions up to 10% and to release encapsulated compounds within human cadaver skin. In vitro release of calcein and BSA from three different encapsulation formulations was measured over time and was shown to be controlled by the encapsulation method to achieve release kinetics ranging from hours to months. Release was modeled using the Higuchi equation with good agreement (r² ≥ 0.90). After microneedle fabrication at elevated temperature, up to 90% of encapsulated BSA remained in its native state, as determined by measuring effects on primary, secondary, and tertiary protein structure. Conclusions Biodegradable polymer microneedles can encapsulate drug to provide controlled-release delivery in skin for hours to months.     

Microinfusion Using Hollow Microneedles

Purpose The aim of the study is to determine the effect of experimental parameters on microinfusion through hollow microneedles into skin to optimize drug delivery protocols and identify rate-limiting barriers to flow. Methods Glass microneedles were inserted to a depth of 720–1080 μm into human cadaver skin to microinfuse sulforhodamine solution at constant pressure. Flow rate was determined as a function of experimental parameters, such as microneedle insertion and retraction distance, infusion pressure, microneedle tip geometry, presence of hyaluronidase, and time. Results Single microneedles inserted into skin without retraction were able to infuse sulforhodamine solution into the skin at flow rates of 15–96 μl/h. Partial retraction of microneedles increased flow rate up to 11.6-fold. Infusion flow rate was also increased by greater insertion depth, larger infusion pressure, use of a beveled microneedle tip, and the presence of hyaluronidase such that flow rates ranging from 21 to 1130 μl/h were achieved. These effects can be explained by removing or overcoming the large flow resistance imposed by dense dermal tissue, compressed during microneedle insertion, which blocks flow from the needle tip. Conclusions By partially retracting microneedles after insertion and other methods to overcome flow resistance of dense dermal tissue, protocols can be designed for hollow microneedles to microinfuse fluid at therapeutically relevant rates.     

Bioceramic microneedles with flexible and self-swelling substrate

To reduce the effort required to penetrate the skin and optimize drug release profiles, bioceramic microneedle arrays with higher-aspect-ratio needles and a flexible and self-swelling substrate have been developed. Swelling of the substrate can assist in separating it from the needles and leave them in the skin as a drug depot. The preparation procedures for this bioceramic microneedle are described in the paper. Clonidine hydrochloride, the model drug, was released in a controlled manner by the microneedle device in vitro. Results showed that the microneedle array with a flexible and self-swelling substrate released the drug content faster than the array with a rigid substrate. Disintegration of the needle material and diffusion of the drug molecules are believed as the main control mechanisms of the drug release from these microneedle arrays. Ex vivo skin penetration showed that they can effectively penetrate the stratum corneum without an extra device. This work represents a progression in the improvement of bioceramic microneedles for transdermal drug delivery.     

Microneedle-assisted delivery of verapamil hydrochloride and amlodipine besylate

The aim of this project was to study the effect of stainless steel solid microneedles and microneedle rollers on percutaneous penetration of verapamil hydrochloride and amlodipine besylate.Verapamil, 2-(3,4-dimethooxyphenyl)-5-[2-(3,4 dimethoxyphenyl)ethyl-methyl-amino]-2-propan-2-yl-pentanenitrile is a calcium channel blocker agent that regulates high blood pressure by decreasing myocardial contractilty, heart rate and impulse conduction. Amlodipine, (R, S)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1, 4-dihydropyridine, is a calcium channel blocker that is used for the management of hypertension and ischemic heart disease. Passive penetration of verapamil and amlodipine across the skin is low. In vitro studies were performed with microneedle-treated porcine ear skin using vertical static Franz diffusion cells (PermeGear, Hellertown, PA, USA). The receiver chamber contained 5 ml of PBS (pH7.4) and was constantly maintained at 37 °C temperature with a water circulation jacket. The diffusion area of the skin was 1.77 cm². The donor compartment was loaded with 1 ml of the solution containing 2.5 mg/ml of amlodipine besylate. The donor chamber was covered with parafilm to avoid evaporation. Passive diffusion across untreated porcine skin served as control. Aliquots were taken every 2 h for 12 h and analyzed by liquid chromatography–mass spectrometry. Transcutaneous flux of verapamil increased significantly from 8.75 μg/cm²/h to 49.96 μg/cm²/h across microneedle-roller treated porcine skin. Percutaneous flux of amlodipine besylate following the use of stainless steel microneedles was 22.39 μg/cm²/h. Passive flux for the drug was 1.57 μg/cm²/h. This enhancement of amlodipine flux was statistically significant. Transdermal flux of amlodipine with microneedle roller was 1.05 μg/cm²/h in comparison with passive diffusion flux of 0.19 μg/cm²/h. The difference in flux values was also statistically significant. Stainless steel solid microneedles and microneedle rollers increased percutaneous penetration of verapamil hydrochloride and amlodipine besylate. It may be feasible to develop transdermal microneedle patches for these drugs.     

Feasibility of microneedles for percutaneous absorption of insulin.

Insulin loaded microneedles were prepared using dextrin as the base for the percutaneous administration of insulin. Under room temperature, insulin solution was added to high concentration of dextrin solution, glue, and microneedles were prepared by forming thread with polypropylene tips. The mean weight of the microneedles was 0.59+/-0.01 (S.E.) mg. The mean length and basal diameter were 3.24+/-0.16 and 0.55+/-0.03 mm, respectively. Five microneedles were percutaneously administered to mice at the insulin dose levels of 0.5, 1.0 and 2.5IU/kg. After administration, blood samples were collected for 5 h and plasma glucose levels were measured. Lowest plasma glucose level appeared at 1 h after the administration of microneedles and dose-dependent hypoglycemic effect of insulin was clearly observed in those dose range. By comparing the mean area above the plasma glucose level versus time curve (AAC) between microneedle preparation and i.v. solution, the pharmacological availabilities were calculated to be 97.7% (0.5IU/kg), 93.3% (1.0IU/kg) and 91.3% (2.5IU/kg), respectively. When highly loaded insulin loaded microneedle was administered to mice with one microneedle, there was not a significant difference on the plasma glucose level versus time curves between 5 and 1 microneedle experiments. In vitro release study showed that almost all of the formulated insulin was released within 1 h. The T50% was estimated to be 15.4+/-1.1 min. Stability of insulin in the microneedle preparations showed that the remaining insulin after 1 month of the storage were 98.2% (-80 degrees C), 98.9% (20 degrees C) and 99.0% (40 degrees C). Evans blue (EB) loaded microneedles were also prepared and histological study was performed with HWY-Slc hairless rats. The diffusion of EB from the microneedle to the environmental skin reached to the maximum at 3 h after administration. The scab was formed at 24 h after administration. The wound formed by the administration of microneedle was cured at 72 h after administration. Those results suggest the usefulness of a self-dissolving microneedle for the percutaneous delivery of peptide/protein drugs like insulin.     

Macromolecular delivery into skin using a hollow microneedle

The objective of the present study was to obtain information to develop an effective delivery device regarding a sophisticated hollow microneedle array-patch system. Thus, the potential of hollow microneedles was investigated for enhancing the transdermal delivery of hydrophilic large molecular compounds, and the effect of variable parameters on drug release behavior was determined from skin. Fluorescein isothiocyanate (FITC)-dextrans (4.3 kDa), FD-4, was used as the main model compound, and it was successfully loaded into the lower epidermis as well as the superficial dermis of the skin in hairless rats by a hollow microneedle. The higher the volume of FD-4 solution injected, the faster the FD-4 release rate from skin. In addition, release rate tended to increase when FD-4 was administered dividedly by multiple injections. These release profiles of FD-4 were expressed by Fick's law of diffusion. Furthermore, a combination of the formulation strategy and hollow microneedle-assisted delivery was useful for controlling the drug release rate from skin. Release profiles from drug-loaded skin were also compared by changing the molecular weights of model compounds. The larger molecular size of compounds caused a lower release rate from skin. These results suggest the utilization of hollow microneedle to enhance transdermal delivery of large molecular compounds and provide useful information for designing an effective hollow microneedle system.     

Microneedles for transdermal drug delivery

The success of transdermal drug delivery has been severely limited by the inability of most drugs to enter the skin at therapeutically useful rates. Recently, the use of micron-scale needles in increasing skin permeability has been proposed and shown to dramatically increase transdermal delivery, especially for macromolecules. Using the tools of the microelectronics industry, microneedles have been fabricated with a range of sizes, shapes and materials. Most drug delivery studies have emphasized solid microneedles, which have been shown to increase skin permeability to a broad range of molecules and nanoparticles in vitro. In vivo studies have demonstrated delivery of oligonucleotides, reduction of blood glucose level by insulin, and induction of immune responses from protein and DNA vaccines. For these studies, needle arrays have been used to pierce holes into skin to increase transport by diffusion or iontophoresis or as drug carriers that release drug into the skin from a microneedle surface coating. Hollow microneedles have also been developed and shown to microinject insulin to diabetic rats. To address practical applications of microneedles, the ratio of microneedle fracture force to skin insertion force (i.e. margin of safety) was found to be optimal for needles with small tip radius and large wall thickness. Microneedles inserted into the skin of human subjects were reported as painless. Together, these results suggest that microneedles represent a promising technology to deliver therapeutic compounds into the skin for a range of possible applications.     

A method to improve the efficacy of topical eflornithine hydrochloride cream

Abstract

Context: Facial hirsutism is a cosmetic concern for women and can lead to significant anxiety and lack of self-esteem. Eflornithine cream is indicated for the treatment of facial hirsutism. However, limited success rate and overall patient's satisfaction, even with a long-term and high-frequency application, leave room for improvement.

Objective: The objective of this study is to test the effect of microneedle treatment on the in vitro skin permeation and the in vivo efficacy of eflornithine cream in a mouse model.

Materials and method: In vitro permeation study of eflornithine was performed using Franz diffusion cell. In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically.

Results and discussion: The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles.

Conclusion: The integration of microneedle treatment into topical eflornithine therapy represents a potentially viable approach to increase eflornithine's ability to inhibit hair growth.     

Dissolvable microneedle patches for the delivery of cell-culture-derived influenza vaccine antigens

Microneedle patches are gaining increasing attention as an alternative approach for the delivery of vaccines. In this study, a licensed seasonal influenza vaccine from 2007 to 2008 was fabricated into dissolvable microneedles using TheraJect's microneedle technology (VaxMat). The tips of the microneedles were made of antigens mixed with trehalose and sodium carboxymethyl cellulose. The patches containing 15 μg per strain of the influenza antigen were characterized extensively to confirm the stability of the antigen following fabrication into microneedles. The presence of excipients and very low concentrations of the vaccine on the microneedle patches made it challenging to characterize using the conventional single radial immunodiffusion analysis. Novel techniques such as capture enzyme-linked immunosorbent assay and enzyme digestion followed by mass spectroscopy were used to characterize the antigens on the microneedle patches. The in vivo studies in mice upon microneedle administration show immunogenicity against monovalent H1N1 at doses 0.1 and 1 μg and trivalent vaccine at a dose of 1 μg. The initial data from the mouse studies is promising and indicates the potential use of microneedle technology for the delivery of influenza vaccine.     

Current aspects of formulation efforts and pore lifetime related to microneedle treatment of skin

Importance of the field: The efficacy of microneedles in the area of transdermal drug delivery is well documented. Multiple studies have shown that enhancement of skin permeation by means of the creation of microscopic pores in the stratum corneum can greatly improve the delivery rates of drugs. However, skin pretreatment with microneedles is not the only factor affecting drug transport rates. Other factors, including drug formulation and rate of micropore closure, are also important for optimizing delivery by this route.

Areas covered in this review: This review aims to highlight work that has been done in these areas, with an emphasis on drug formulation parameters that affect transdermal flux.

What the reader will gain: This review creates an appreciation for the many factors affecting microneedle-enhanced delivery. Most results clearly indicate that microneedle skin pretreatment by itself may have different effects on drug transport depending on the formulation used, and formulation characteristics have different effects on the transport through untreated skin and microneedle-treated skin. Several formulation approaches are reported to optimize microneedle-enhanced drug delivery, including co-solvent use, vesicular, nanoparticulate and gel systems.

Take home message: In addition to well-established factors that affect microneedle-assisted delivery (geometry, type of microneedle, etc.), formulation and pore viability are also critical factors that must be considered.     

可分离微针研究进展

可分离微针属于可溶性微针的一种,应用于皮肤后其针尖与背衬可快速分离,有效减少了微针的佩戴时间以及提高了给药效率,是一种新型透皮给药系统,因此近年来成为研究热点。目前国内外已经开发出多种形式的可分离微针,体内外研究证实可分离微针具有广阔的应用前景。本文概述了当前可分离微针的特点与分类,以期对后续可分离微针的开发与应用提供参考。     

胰岛素相转化水凝胶微针透皮贴片

本研究采用化学惰性、可吸收体液溶涨从玻璃态变为水凝胶态的聚合物材料制备了可用于胰岛素透皮给药的微针贴剂,相转化水凝胶微针贴膜。胰岛素是1型和晚期2型糖尿病的必用药。针尖穿透表皮后吸收体液溶涨,使预先担栽的胰岛素在真皮层高效而精确地释放出来,同时不留下针尖物质在皮层的沉积。上述性质使得相转化水凝胶微针贴膜成为需要一生频繁用药的蛋白、多酞药物的理想剂型。     

Effect of microneedle on the pharmacokinetics of ketoprofen from its transdermal formulations

Non-invasive transdermal delivery using microneedle arrays was recently introduced to deliver a variety of large and hydrophilic compounds into the skin, including proteins and DNA. In this study, a microneedle array was applied to the delivery of a hydrophobic drug, ketoprofen, to determine if transdermal delivery in rats can be improved without the need for permeation enhancers. The ability of a microneedle to increase the skin permeability of ketoprofen was tested using the following procedure. A microneedle array was inserted into the lower back skin of a rat using a clip for 10 min. Subsequently, 24 mg/kg of a ketoprofen gel was loaded on the same site where the microneedle had been applied. Simultaneously, the microneedle was coated with 24 mg/kg of a ketoprofen gel, and inserted into the skin using a clip for 10 min. As a negative control experiment, only 24 mg/kg of the ketoprofen gel was applied to the shaved lower back of a rat. Blood samples were taken at the indicated times. The plasma concentration (C_p) was obtained as a function of time (t), and the pharmacokinetic parameters were calculated using the BE program. The group loaded with the microneedle coated with ketoprofen gel showed a 1.86-fold and 2.86-fold increase in the AUC and C_max compared with the ketoprofen gel alone group. These results suggest that a microneedle can be an ideal tool for transdermal delivery products.     

新型经皮传递胰岛素透明质酸微针制剂的制备及性能考察

目的证明透明质酸微针制剂在药物经皮传递系统方面的应用前景。方法通过皮肤及微针的显微照片考察微针刺入皮肤的性能和在大鼠体内的溶解性能;用皮肤刺激性实验评价透明质酸微针的安全性;以人的离体皮肤为透皮释药模型,通过体外经皮通透实验考察微针对模型药物胰岛素经皮吸收的促进作用。结果微针能够均匀刺穿角质层,在皮肤表面产生与微针一致的阵列形状,在皮肤断面可观察到直至真皮层的通道;在大鼠体内使用1 h后,针体能够完全溶解,皮肤刺激性指数为1.7,属于轻度刺激性;体外经皮实验中,微针中的胰岛素能够以活性形式释放,与同剂量的溶液相比,微针对胰岛素的体外经皮吸收具有显著的促进作用,稳态通透速率达75.33×10-6U.cm-2.h-1。结论以透明质酸为基质制备的微针具有良好的皮肤刺入性、溶解性和轻度的刺激性,对于生物大分子类药物的经皮吸收有明显的促进作用,具有良好的开发前景。     

滚轮微针处理次数对药物皮肤渗透、分布及皮肤刺激性的影响

目的 考察用于皮肤促透研究的滚轮微针处理次数。方法 以维A酸为模型药物,Franz扩散池、组织匀浆法研究裸鼠皮肤的促透效果,用亚甲蓝染色法、激光共聚焦显微镜法等考察裸鼠皮肤的药物分布,经皮水分流失（TEWL）测量法、激光多普勒血流量法评价皮肤刺激性。结果 滚轮微针处理次数为1、3、5、8、10次时,处理次数越多,促透效果越好,皮肤中滞留药量也越高,但是皮肤中滞留药量在处理8次和10次时无显著性差异（P>0.05）。亚甲基蓝染色和激光共聚焦实验均显示处理次数增加,针孔分布均匀度提高;亚甲基蓝染色显示,处理5次以上时,出现部分针眼重叠而皮肤破损现象。在体皮肤内药物吸收实验显示处理1次时,裸鼠皮肤中滞留药量均匀性较差,但是处理3次以上时,皮肤中滞留药量均匀性明显改善。TEWL测量法显示处理5次以下,皮肤屏障功能恢复时间为24 h,处理8次以上为36 h。激光多普勒血流量法显示处理5次以下,皮肤屏障功能恢复时间为1 h,处理8次以上为2 h。结论 滚轮微针处理5次可确保裸鼠皮肤促透研究的安全性和有效性。     

Influence of the delivery systems using a microneedle array on the permeation of a hydrophilic molecule, calcein.

Despite the advantages of drug delivery through the skin, such as easy accessibility, convenience, prolonged therapy, avoidance of the liver first-pass metabolism and a large surface area, transdermal drug delivery is only used with a small subset of drugs because most compounds cannot cross the skin at therapeutically useful rates. Recently, a new concept was introduced known as microneedles and these could be pierced to effectively deliver drugs using micron-sized needles in a minimally invasive and painless manner. In this study, biocompatible polycarbonate (PC) microneedle arrays with various depths (200 and 500mum) and densities (45, 99 and 154ea/cm(2)) were fabricated using a micro-mechanical process. The skin permeability of a hydrophilic molecule, calcein (622.5D), was examined according to the delivery systems of microneedle, drug loading, depth of the PC microneedle, and density of the PC microneedle. The skin permeability of calcein was the highest when the calcein gel was applied to the skin with the 500mum-depth PC microneedle, simultaneously. In addition, the skin permeability of calcein was the highest when 0.1g of calcein gel was coupled to the 500mum-depth PC microneedle (154ea/cm(2)) as well as longer microneedles and larger density of microneedles. Taken together, this study suggests that a biocompatible PC microneedle might be a suitable tool for transdermal drug delivery system of hydrophilic molecules with the possible applications to macromolecules such as proteins and peptides.