Micro/nano needles for advanced drug delivery

Xingchun Meng ,Zeyu Zhngc, ,Linlin Li,b,c,*

a Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience,Beijing Institute of Nanoenergy and Nanosystems,Chinese Academy of Sciences,Beijing,100083,China

b School of Nanoscience and Technology,University of Chinese Academy of Sciences,Beijing,100049,China

c School of Chemistry and Chemical Engineering,Guangxi University,Nanning,530004,China

Keywords:Micro-and nano needle Drug delivery Noninvasiveness Stimuli-responsive

ABSTRACT Along with the boost of drugs’ exploitation,drug delivery has been a common concern among researchers and patients.As an emerging strategy,the micro-and nano-needle(MNN)is a single or an array of micro/nanoneedles,and promises to be a robust technology for efficient,noninvasive and convenient drug delivery.Compared with the conventional injection,MNN can pierce the bio-membranes to transfer cargos without any damage of tissue or nerves.And regarding the topical drug release,MNN is more efficient than oral administration and topical cream.More specifically,microneedle,a powerful technique,has been widely applied in different diseases such as diabetes,anti-obesity and contraception through skin and ocular surface.Correspondingly,many novel microneedles and optimization methods have been developed recently to improve the drug delivery efficacy.Nanoneedle,much smaller than microneedle,can minimize the skin damages caused by the repeated use of microneedles.Nanoneedles with nanoscaled size are usually used to penetrate the cell membrane.And recently many stimulusaided methods like electrical and magnetic force have been invented to deepen penetration and improve transfer effects.The aim of this review is to introduce the recent advances of MNN-based drug delivery,including their types,application locations and diseases,optimization of delivery strategies and future development prospects.*Corresponding author.

1.Introduction

Drug delivery has been a hotpot with the rapid development of modern medicine,and plenty of delivery methodologies,such as oral administration,injections and ointment have been applied over the past decades[1,2].The rational selection of one specific technology depends on the drug's unique pharmacological properties,so that we can make full use of the drug's therapeutic effects and lower its side effect in the clinic.However,each of these drug administration methods have their own advantages and disadvantages.Oral administration is common and simple in daily life,but it is inconvenient for some special human populations.And it has low bioavailability due to the hepatic first-pass elimination and gastrointestinal decomposition [3,4].Hypodermic,intramuscular and intravenous injection can transfer the drug molecules into the subcutaneous tissue and vessels,but it may cause pain and tissue damage.The injections also put high requirements for drugs such as good water solubility [5–7].Another drug administration is transdermal delivery like topical cream that is a traditional way and easy to operate.However,the stratum corneum of human skin is a tough physical barrier towards chemical environments,restricting drug's permeation and effects [8–10].

Therefore,it is urgent to advance existing drug delivery strategies and find safe,efficient and cost-effective operations.Either modifying “free”drugs with particulate carriers or improving the delivery technology is feasible [11–14].Compared with molecular-level chemical modification,the latter is much more convenient and valuable,since it provides a common technology for various kinds of drugs.According to this design concept,the researchers have improved conventional transcutaneous drug delivery and invented a novel strategy:micro-and nano-needle (MNN)[15,16].It is a burgeoning family of advanced drug delivery technology,bringing favorable drug's pharmacokinetics as well as good patient compliance.MNN is a single or an array of micro-or nano-scaled needles with high-aspect-ratio structures.They are capable of overcoming the barrier of membrane or stratum corneum,and releasing drugs into systemic circulation with high efficiency.This novel technology is portable and controllable,offering a facial platform for various drug delivery [17–23].

Herein,we summarize advanced drug delivery strategies employing micro-and nano-needles,and review their advances including types of MNN,function mechanisms and applications in diabetes,obesity,hair loss,contraception and cancer.Recent progresses in these fields are emphatically discussed.We also compare the respective advantages of microneedles and nanoneedles and discuss their state-of-art research results and relationship.Finally,we propose the future challenges in the development and clinical translation of MNN.

Fig.1.Illustration of different types of microneedles and their applications in skin and ocular surface.(a)Two types of microneedles including traditional microneedles (A– Solid MNs;B– Coated MNs;C–Hollow MNs) and emerging microneedles (D–Dissolving MNs;E -Hydrogel forming MNs;F–Backward-facing barbed MNs [43] Copyright 2020 John Wiley and Sons and Clamping MNs [44]Copyright 2019 Elsevier);Microneedles were applied in (b) skin [9] (Copyright 2015 Elsevier) and (c)ocular surface[45](Copyright 2015 John Wiley and Sons).All figures were adopted with permission.

2.Microneedles

2.1.Types of microneedles

Microneedle (MN) is the miniaturization of a single or an array of needles with micrometer-scale length.It can penetrate human stratum corneum and produce micron-sized pathway for facilitating drugs’ diffusion and function in a noninvasive and efficient way.With technology advancing,researchers have created various fabrication methods including photolithography,laser cutting,micro-electromechanical systems and chemical etching to make different kinds of microneedles[24–26].These MNs can be roughly classified into two different groups:traditional MNs (including solid MNs,coated MNs and hollow MNs)and emerging MNs (including dissolving,hydrogel-forming and specially-structural microneedles) (Fig.1).

2.1.1.Traditional microneedles

Solid microneedles (solid MNs),generally made of metal [27],silicone[19]and insoluble polymers[28]are the first to be applied in the osmotic penetration area.Solid MNs are used to penetrate skin to produce temporary micro-channels,and then the drugs added on the skin surface can reach dermis and enter the blood circulation to exert pharmacological effects.The limitation is that this process is cumbersome and troublesome,and the rapid recovery of the skin-carrying channels limits the wide application of solid MNs.

As an improved version,coated microneedles (coated MNs) are surrounded with a drug layer on the basis of solid MNs[29–32].While piercing the skin,the coating drugs are simultaneously released into the blood and interstitial fluid to take effect.This method can be administered in one step with higher efficacy than the solid MNs.However,the actual drug loading is usually low due to the limited thickness of coating layer and MNs’ size [33].

Compared with solid MNs and coated MNs,hollow microneedles(hollow MNs) are syringe needle -like and can realize a large dose and continuous drug release [34–36].With a hole at tip and an empty space inside,the drug solution can not only continuously reach dermis layer and play pharmacodynamics,but can also implement in a controllable way through adjusting drug-flow rate and concentration.What needs to be solved is that the micropores are easily to be blocked during inserting into the tissue,which seriously affects subsequent delivery of drugs.

2.1.2.Emerging microneedles

During the recent several years,the researchers have advanced the existing technologies and developed many new MNs with complex structures,including dissolving,hydrogel-forming and specially-constructed microneedles to improve the performance.Dissolving microneedles (Dissolving MNs) are prepared from biodegradable polymers with drugs encapsulated inside [37,38].After piercing into the tissue,the body of the microneedles would dissolve and release loading drugs in a controllable way.This process is one single step and not needed to remove needles,which is favorable to long-term therapy and increase patient compliance.For instance,Prausnitz and colleagues encapsulated biomolecules(sulforhodamine B,bovine serum albumin,and lysozyme) within biocompatible carboxymethylcellulose or amylopectin formulations to fabricate dissolving microneedles.When inserted into skin,the microneedles could dissolve within the skin to achieve a bolus or sustained biomolecules’ delivery,which would not leave behind any biohazardous sharp waste[39].

Hydrogel-forming microneedles (Hydrogel-forming MNs) are fabricated with super-swelling cross-linked polymers with drugs loaded inside[40–42].When accessing interstitial fluid of tissue,the hydrophilic structure of the microneedles can absorb water and swell greatly,so that the loaded drugs can be released into the circulation quickly.Donnelly et al.designed hydrogel-forming microneedles to sustainably transfer drug metformin HCl transdermally in a high dose [41].Once applied into the skin,MNs can be in contact with interstitial fluid to swell and create porous aqueous micro-conduits to diffuse drugs into dermal microcirculations.

In addition,many specially-constructed microneedles have been designed to improve the loading rate and delivery efficacy of drugs.For instance,Lee's group has also presented specialized microneedles with backward-facing curved barbs [43].It was found that these special microneedles showed 18-times stronger tissue adhesion than the barbless MNs,which is a robust performance for sustained and stable drug release.Zhao et al.have reported serration-like clamping microneedles by means of ferrofluid-configured moldings [44].This array was capable of adhering onto the tissue firmly,resulting in a long-time function even when recipients were exercising.

Fig.2.Microneedles' developments in different diseases.Microneedles were explored in (a) obesity [60] (Copyright 2017 John Wiley and Sons),(b) hair loss [65](Copyright 2019 American Chemical Society) and (c) cancer [71] (Copyright 2017 The American Association for the Advancement of Science).All figures were reproduced with permission.

2.2.Microneedles for advanced drug delivery

2.2.1.Microneedles in skin and ocular surface

Skin is the largest organ of human body,about 2 m2of surface.It has many vital functions (endocrine,thermoregulation,sensory etc.),and can effectively protect us from external chemical and physical assaulters [9,10,46–48].Mammalian skin is consisted of 2 main layers:the outermost layer epidermis and the innermost layer dermis.Composed of five layers,the epidermis has a thickness varying from 50 to 150 μm(palms and soles excluded)depending on different regions.The dermis mainly comprising papillar and reticular layer has many active sites.The papillar layer is connected with epidermis and is rich in vessels and nerve endings,while the reticular layer containing fibrous bundles supports many vital functions like immunologically active cells,blood capillaries,nerves,eccrine and apocrine glands.Rigid needles with large length can reach deep region of skin like dermis and hypodermic to release drugs,but might damage the tissue and nerves to cause pain.In contrast,the length of a microneedle is kept only in microscale (length=200–2000 μm,aspect ratio (AR)=0.7–100).After piercing into the skin surface,it would create temporary conduits between few microns,and help drug overcome barrier of stratum corneum and diffuse into the vessels without damaging nerves.Compared with systematic injection treatment,microneedle-based drug delivery system can locally work on targeted area and control drug release accurately.Importantly,this process could realize self-administration by patients after simple training,especially for long-term drug release for the treatment of many chronic diseases.

Eye is an important organ.It becomes more fragile with the increase in aging population,wear of contact lens and eye diseases worldwide.Systematic drug delivery(parenteral or oral route) requires high dosage to ensure locally efficient concentration,while conventional topical drug delivery (drops or cream) is simple but has low bioavailability (＜5%absorption) and fast to clean.Therefore,it is still an unmet medical demand for long-lasting,localized and efficient ocular drug delivery with good patient compliance [45,49–51].However,this process is challenging because of eye's complex structure.Especially,cornea where most injuries or diseases occur lacks blood vessels and is nourished by tears and aqueous humor.The protection from continuous eyelid blinking and fluid turnover is also a major barrier for drug delivery[50,51].In 2007,Prausnitz and colleagues first raised coated microneedle for ocular drug delivery[52].The microneedles(500 μm in length and 45° in tip angle)were rigid enough to pierce human cadaveric,and drug coating dissolved quickly (＞30 s) from the needles to reach cornea or sclera tissue.It is reported that fluorescein-coated microneedles released 60 times higher fluorescein concentrations than conventional topical administration.Similarly,Chen et al.reported a new double-layered microneedle patch to control drug delivery to eyes painlessly [51].In corneal neovascularization model,these microneedles with a MeHA outer layer and a HA inner core could insert into ocular tissue and act as micro-drug-reservoirs(e.g.anti-angiogenic monoclonal antibody)to work.It is shown that the eye path produced significant decline (～90%) in neovascular area,which was enhanced by anti-inflammatory compounds (diclofenac).Such microneedle-based ocular drug delivery provides a cost-effective,localized and high-efficient strategy.It promises painless and homebased therapy of many ocular diseases.

Besides skin and ocular drug delivery,microneedle array is also capable of transferring biomolecules and drugs to other tissues and organs such as vascular tissue,gastrointestinal tract and genitourinary system in an implantable way.These afore-mentioned non-transdermal drug delivery cases have been introduced extensively in previous review [15,20,53,54],so we do not discuss too much here.

2.2.2.Microneedles for different diseases

Microneedle-based delivery can be applied on topical lesion locations and plays a role in a high-efficiency and noninvasive therapy,avoiding complications that might be brought by systematic administration therapeutics (Fig.2).So,this method has captured plenty of attention and been applied in many fields including blood glucose control [55–59],obesity [60–63],hair loss [64,65],contraception [66,67],as well as cancer [68–71].In 2015,Gu et al.first presented a microneedle-based insulin delivery system and achieved smart regulation of blood glucose in mouse models with type 1 diabetes[59].The key to the device is the fast release of encapsulated insulin in a hypoxic state caused by glucose's enzymatic oxidation in the hyperglycemic environment.This fast-responsive,ease-administrative and painless device was more sensitive than commonly-used injection and laid a foundation for intelligent mediation of blood glucose.Chen's group has designed a polymeric microneedle patch to transfer anti-obesity compounds to overcome obesity(Fig.2a) [60].Slowly dissolving microneedles delivered drugs to subcutaneous calorie-storing white adipose and transformed it into calorieburning brown one to raise energy consumption,suppressing the gain of fat tissue.Such microneedle arrays realized a long-term home-based administration in a lower effective dosage and didn't need daily government.Gu's group also created a novel microneedle patch to solve hair loss problems(Fig.2b)[65].They created an activator deliver system to sustainably transfer follicle stem cells to promote hair follicle regrowth.It was reported that this microneedle device,mainly made of hair-derived keratin,could obviously improve the therapeutic effect with a minimal dose,leading to augmented pigmentation and hair regrowth in a mouse model within 6 days.A continuous contraceptive-releasing microneedle array has been also reported[67].Microneedles,made of biodegradable polylactic acid and polylactic-co-glycolic acid could pierce the skin effectively and then snap offwithin 5 s to achieve a sustained release of levonorgestrel.This device was successfully conducted in the rat models and provided a long-acting contraception conception for women.Meanwhile,a melanin-mediated microneedle patch was developed to achieve cancer immunotherapy.Melanin is able to transform almost all sunlight energy absorbed (99.9%) into heat.Under near-infrared irradiation,melanin in the patch could generate local heat and promote dendritic cells' uptake of the tumor antigen,enhancing T cells' activity through vaccines (Fig.2c) [71].This kind of spatiotemporal photo-responsive immunotherapy significantly improved the antitumor immune effects towards primary tumor and distant tumor in mice.Due to the slow release characteristics of local administration,the microneedles have broad application prospects in the treatment of chronic diseases,showing great advantages over traditional drug delivery methods.

2.2.3.Smart stimuli-responsive drug delivery

The existing microneedle arrays for drug delivery are mostly administered in a “passive” way where the transfection efficiency is restricted by slow diffusion of drugs from needles,leading to potential undesirable problems like uncontrollable release,slow effect or drug overdoses.In order to solve these problems,autonomous dynamic delivery strategy has been invented based on the stimuli-responsive ability and provided broad prospects for therapeutic applications.Triggered by exogenous(heat,force and electricity,etc.)[35,57,59,72–77]and endogenous(such as glucose and thrombin) stimuli [59,78,79],microneedles can accurately release drugs in the targeted region quickly in a versatile and effective way,achieving an intelligent controlled release (Fig.3).

Ultrasound and iontophoresis are also conductive to the drug absorption.When employed in skin,ultrasound can induce skin's changes(vibration,contraction and relaxation) and thermal effects,while iontophoresis can accelerate ionized biomolecules' penetration towards tissue in the electric field.Based on this conception,Lim et al.developed hyaluronic acid (HA) microneedles for active local delivery(Fig.3a) [72].Ultrasonication causes microneedles' dissolution,and alternating current (AC) iontophoresis simultaneously promotes the moving of HA ions and ionized drugs by means of electrostatic force.This cooperation between ultrasound and iontophoresis greatly shorten reaction duration and speed up biomolecule diffusion.Gas can help break the dermal barriers of skin and promote the drug delivery effectively.Based on this,Wang's group designed an autonomous microneedle delivery system utilizing magnesium microparticles as an inner engine for intradermal payload delivery (Fig.3b) [77].Once patch piercing into the skin,the loaded magnesium microparticles was responsive with interstitial fluids and produced H2bubbles rapidly,opening skin channels and facilitating quick release of cargos.This method was applied in the in vivo experiments to transfer anti-CTLA-4 and obviously boosted the immunity response and survivals.Mechanical force is an easily accessible stimulus to active and spatiotemporallycontrolled microneedle drug delivery.Gu's group reported a straintriggered drug transfer system comprising stretch-sensitive drug-loaded unit and microneedles(Fig.3c)[74].When a tensile strain was applied on the device,the microdepot generated Poisson's ratio-induced compression and enlarged diffusion surface,leading to the rapid drug release.This device was successfully used in the transdermal insulin delivery and blood glucose's regulation in type 1 diabetic mouse models.Heat is also an important factor for quick drug release especially for thermo-sensitive materials.Based on thermo-responsive microneedle patch,Kim et al.designed an integrated system for closed-loop,realtime diabetes therapy[57].This system comprised a stretchable device equipped with gold mesh and gold-doped graphene,polymeric microneedles and electrical components like heater,and temperature,glucose and pH sensors.High glucose concentration recorded by synergy of various sensors could activate heaters to melt PCM,and then bioresorbable microneedle array released metformin to the targeted area as a feedback to regulate glucose levels.

Smart microneedle systems administrated by endogenous stimuli have been invented to control drug release and diffusion automatically.In medical pathology,this system can be activated by physiological responses(glucose,thrombin)for one specific disease,such as diabetes and thrombosis[59,78,79].Diabetes whose typical symptom is blood glucose imbalance is a common disease,and needs timely blood glucose monitor and insulin injections.As we discussed before,Gu et al.explored a new enzyme-based glucose-responsive “closed-loop” insulin release microneedles(Fig.3d)[59].It was fabricated with painless MNs and hypoxiasensitive HA vesicles with insulin inside.While piercing into the skin of patients,the hyperglycemic state would stimulate the glucose enzymatic oxidation to cause local hypoxic environment,contributing to the vesicles' quick dissociation and encapsulated insulin's precise release.Utilizing this cooperation between MNs and stimuli-responsive drug carriers,Gu's group extended the therapeutics and reported a feedbackcontrolled anticoagulation MNs patch with noninvasiveness and minimal risk of under-or over-dosage [79].The thrombin cleavable peptide was introduced as a linker between heparin (a common anticoagulant) and HA chain,and the thrombin's activation in body could cleave this peptide,thus triggering heparin release automatically.This device was then used in the thrombolytic challenge model and it was shown that it effectively protected mice from acute pulmonary thromboembolism in a long-term manner.As a consequence,compared with traditional MNs,the emerging stimuli-responsive microneedle system provides an autonomous dynamic delivery strategy to transfer biomolecules on demand,so that the drugs can be released in the targeted areas through a high-efficient and smart way.

3.Nanoneedles

Although microneedle arrays play vital role especially in transcutaneous drug delivery,their repeated use could cause skin irritation to a certain extent.Nanoneedles(NNs),however,do better in this aspect,as they have the same working principle as MNs,but with much smaller,about nanometersize (sub-100 nm diameter).They could pierce cell membrane and transfer biological cargos into cell interior.Obviously,this penetration process perturbs cells in a minimal way and cells tend to keep good healthy post-delivery [80–84].Compared with conventional microinjection and electroporation,NNs provide a more efficient,easy-to-use and precise method of delivering cargoes with minimal damage and high throughput.The micro-and nano-needles have their own advantages and disadvantages,but few reviews have put them together for summarization [85].

3.1.Nanoneedles for advanced drug delivery

Nanoneedle with high-aspect ratio nanostructure inherits prominent mechanical,electrical,optical properties,and is able to active cytosolic delivery,coordinate cell membrane and endo/lysosomal organization to achieve high-efficient nano-therapeutics (Fig.4).Nanoneedle can be fabricated by chemical synthesis [86,87] and nanofabrication (directwrite nanofabrication[88]and focused ion beam machining[89]).It is a painless and powerful way for molecular delivery,cell manipulation,gene transfection and bio-sensing,and significant advances have been made in these fields during this decade [16,80].Nucleic acid delivery towards targeted muscle regions remains difficult and low-efficient because of low transfection efficiency,questionable safety and poor scalability among different cells or locations.Given this problem,Tasciotti's group invented a biodegradable and tunable nanoneedle array for delivering nucleic acids with high efficacy,limited toxicity and practicability without complex equipment [90].It was prepared via chemical etching of silicon and could traverse the cytosol to simultaneous transfer siRNA and DNA with high efficiency (＞90%).Meanwhile,this nanoneedle array was used to transfect VEGF-165 gene,contributing to sustainable neovascularization and 6-folds rise in blood perfusion in specific tissue.Lee's group improved traditional all-siliconbased technology and developed a new strategy of constructing vertically-ordered Si nanoneedles on a thin elastomer layer to achieve mechanical flexibility and optical transparency (Fig.4a)[91].They successfully realized biomolecules' intradermal and intracellular nanoinjection into living cells and tissues.

Fig.3.Smart stimuli-responsive drug delivery based on microneedles.Autonomous dynamic delivery strategy has driven by exogenous stimuli like (a) Ultrasound and iontophoresis [72] (Copyright 2020 Springer Nature),(b) gas [77] (Copyright 2019 John Wiley and Sons) and (c) strain [74] (Copyright 2015 American Chemical Society),and driven by endogenous stimuli like(d)glucose[59]((Copyright 2015 National Academy of Sciences)and(e) thrombin light[75](Copyright 2016 John Wiley and Sons).All figures were reproduced with permission.

Thanks to the nanometer sizes,the nanoneedles can avoid skin irritation caused by repeated use of microneedles to a certain degree.But in another aspect,nanoneedles face problems of limited tissue accessibility and low deliver efficiency through mechanical penetration alone.It is still difficult to fabricate nanoneedle with high aspect ratio (e.g.,AR ＞10).Even though nanoneedles with high AR could be fabricated,they are brittle with small diameter,difficult to penetrate tissues effi-ciently [16,91,92].Many complimentary approaches such as electrical stimulation and magnetic forces are helpful to the solution [93,94].Electroporation can be used to produce transient pores in plasma membrane to introduce biomolecules into cells.Combining with electroporation,nanoneedles can work as both drug reservoirs and electrodes with enhanced localized electrical fields in pointed tips.Li and colleagues have integrated silicon nanoneedles with triboelectric nanogenerator(TENG) to fabricate a nanoneedles-based self-powered drug delivery device and achieved high-efficiency electroporation drug delivery(Fig.4b)[93].It is noticeable that TENG provided a stable voltage pulse to increase the plasma membrane potential and enhance biomembrane permeability,while silicon nanoneedles created nanoscale channels for biomolecules' transfection and increased nanoneedle–cell interfaces'electrical field for enhanced molecular influx.Compared with passive mechanical penetration (＜23%),TENG-exerted system had four-times higher delivery efficiency without jeopardizing cell vitality.They used this device to transport molecules and siRNA inside primary cells,a kind of hard-to-transfect cells with an efficiency over 90% and cell viability greater than 94%.When applied in the living mouse models,this selfpowered system achieved excellent transdermal drug delivery effect(approximate 4-folds enhancement) through biomechanical actions including finger frictions and hand slapping.Besides electrical fields,magnetic force is a good alternative for high-throughput and precise subcellular delivery.Weiss's group coupled the magnetic forces and nanoneedles to develop a magnetic nickel nanospears for precision-guided intracellular gene delivery without any chemical propellants (Fig.4c)[94].It is shown that nanospears were guided by rotating magnet to achieve single cell targeting and co-transfer of biological molecules,and could also be scaled progressively for massive cells' simultaneous batch processing.After nanospear-mediated transfection,the high transfection rate (＞80%) and cell viability (＞90%) could be realized in parallel,exceeding traditional non-viral intracellular technologies.

3.2.Mechanism of nanoneedle-based drug delivery

Fig.4.Nanoneedles in advanced drug delivery.Nanoneedles were developed in (a) conventional drug delivery [91] (Copyright 2019 American Chemical Society)and novel drug delivery driven by(b)electricity[93] (Copyright 2019 John Wiley and Sons) and(c) magnetics[94](Copyright 2018 American Chemical Society).All figures were reprinted with permission.

It is necessary to understand the nanoneedles-controlled processes so as to better design rational technology and promote the clinical translation[81,82,95].Some studies initially assumed that nanoneedles could penetrate the cell membrane and transfer the bioactive molecules into cytoplasm efficiently.According to the atomic force microscopy data,it is shown that nanoneedles interacting with the lipid bilayer and cell membranes,the force–displacement curve showed obvious drops,which were explained by the breaching of the cell membrane.This viewpoint was supported by the microscopy results of nanoneedle-cell interfaces,which presented intact cells all the time [81,82].A latest report disclosed the principle of biomolecules traversing through nanoneedle-biomembrane interface [95].They used the electron and scanning ion conductance microscopy to visualize the change of lipid bilayers and related proteins to describe the whole biological process.It is shown that nanoneedle arrays can improve the internalization of molecular biomarkers,enhancing the endocytosis mediated byclathrin and caveolae simultaneously,as well as macro pinocytosis,which was conductive to nanoneedle-regulated transfer of nucleic acid inside the stem cells.These phenomena not only deepen the comprehension of the nanoneedle-mediated biological processes and intracellular interactions,but also offer powerful ideas for advanced cell-manipulation technology.

4.Conclusions and challenges

MNN is a micro/nano-sized system which can produce small conduits in biomembranes,overcoming the barriers during drug release and diffusion.It is promising to be a robust technology for efficient,noninvasive and convenient drug delivery.Since first appeared several decades ago,MNN has been progressing dramatically in fabrications,delivery methods,and applications (Fig.5).As for microneedles,a large number of novel designs including dissolving,hydrogel-forming and specially-structure microneedles have been invented to improve the drug loading amount,bioavailability and tissue adhesion,while their targeted sites have been expanded from skin to eyes,as well as broadened disease categories from diabetes,skin cancer to anti-obesity,hair regrowth and contraception.Nanoneedles,with smaller size than microneedles have presented similar advances but have been inclined to carry cargos (nucleic acid and protein) into living cells.Importantly,external stimuli like mechanical and magnetic forces,electric fields and heat can boost cargos’ release and accelerate the diffusion process.

While significant progresses of MNNs mediated drug delivery have been made recently,the challenges of MNN in this area still remain.Foremost,it is the transition from efficient transfer of loaded biomolecules to cells’ activation after operation.This means that minimal dose of drugs,mild deliver conditions and good patient compliance are expected to be realized simultaneously to assure maximal treatment effect with minimal disturbance to cells.Another challenge is the designing and application of smart MNN delivery system,which is complex and remains ongoing research subjects.Integrated with mobile devices,the physiological marks or drug release process is supposed to be continuously monitored and controlled in one specific disease (diabetes,cancer and epilepsy).Clinic translation is the ultimate goal and another unresolved hurdle.There is still a long way to commercialize MNNs,since this process can be limited by many factors,such as existing fabrication technology,storage and transportation of bioactive molecule-coated MNs,and monitoring methods.Luckily,a few MN treatments in combination with other approaches (radio frequency,subcision and fractional skin resurfacing) have reached the market for the treatment of skin rejuvenation,hair growth,wound healing,vaccination and diabetes.As the fabrication technology and medical science advancing,more progresses in MNN will eventually be made to address existing issues.We believe that this review has captured latest progresses about MNN and could encourage more innovations in the near future.

Fig.5.Summary of microneedles and nanoneedles and their applications.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgements

The work was supported by the National Key R&D project from Minister of Science and Technology,China (2016YFA0202703);the National Natural Science Foundation of China (81471784,21701180);and the Nature Science Foundation of Beijing (2172058).