One-dimensional microstructure-assisted intradermal and intracellular delivery
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The advancement in the materials manufacturing at micrometer and nanometer scales has already enabled numerous applications in electronics, optics, chemistry, biology and medicine. Biomedical devices carrying micro-/nanostructures are currently being widely used in drug delivery, drug release, biosensing and therapy. New clinical methods for disease diagnosis and treatments are being developed enabled by nanotechnology. One-dimensional (1D) structures are playing an important role in the direct drug delivery both in vivo and ex vivo among various micro-/nanostructures. Here, in this paper, we reviewed recent progresses made on next-generation intradermal and intracellular delivery strategies and applications with focus on 1D microstructure-based approaches.
KeywordsBiomedical devices Intradermal Intracellular 1D nanostructure Drug delivery
1D microstructure-assisted intradermal delivery
Skin is known as the largest organ in the body; it serves to protect the body from the outside environment. Small molecule-based medicine applied on the skin can diffuse through the skin barrier to the body. However, not all medicine can penetrate through the skin to reach the capillaries. Controlled release of particular drugs to the blood vessels and the desire for drugs to release locally remain as challenging tasks. Among the various recently developed techniques, 1D microneedle-based intradermal delivery is among the most promising next-generation intradermal delivery strategies [18, 19, 20, 21]. Microneedles are 1D sharp structure, which can easily puncture the skin barrier to achieving proper drug penetration.
The precise microfabrication techniques are enabling flexible and customizable dimensions and materials of microneedles for specific applications. Materials for microneedles include semiconductors, metals and polymers [11, 22, 23, 24, 25]. For examples, biodegradable porous silicon microneedle arrays were reported fabricated via combination of conventional photolithography with proper etching techniques. Polymer microneedle arrays of ~ 500 μm in length and ~ 5 μm in tip size were fabricated via a micro-molding process using a laser engineered molds . Among different materials, polymer-based microneedles have several advantages over others. First, polymer microneedles can be manufactured at much lower cost due to scalable replica molding process. Second, polymeric microneedles have lower risk as sharp biowastes [11, 22, 23, 24]. Third, polymers are a group of well-studied matrix materials for drug delivery which can themselves be bioactive [11, 27, 28, 29].
Microneedles have many advantages over the traditional syringes. First, it is a gentle and kid-friendly approach, because the micrometer-sized needles transverse the stratum corneum without stimulating the nerves, thus avoiding causing pain to the patients [11, 30, 31, 32, 33]. Microneedles can be integrated to traditional patches to become microneedle patches. Small injection tasks can be done by the patients, thus reducing the frequency to visit a hospital. The microneedle patch can deliver drug locally at the skin tissue, which can bypasses the systemic circulation, thereby reducing the loss of efficacy and side effects . Microneedle patches can deliver a wide selection of drugs, including proteins, antibodies and vaccines [20, 34, 35, 36]. For example, insulin  and growth hormone  have been successfully delivered through the animal skin using microneedles. Microneedle patches administrated vaccines (such as influenza and hepatitis B) can reach the skin tissue easier while avoid the systemic dosing [25, 38]. In addition, microneedle patches can be used to fight obesity, e.g., the microneedle carried degradable nanoparticles releasing browning agents that can transform white adipose tissue into brown adipose tissue that suppresses weight gain [39, 40].
1D microstructure-assisted intracellular delivery
Passive puncture and passive delivery strategy
As shown in Fig. 3a, cells (usually adherent cells) are directly cultured on the substrate containing nanoneedles preloaded with biomolecular cargo. The cargo dissociates from the nanoneedles upon physical penetration through the cellular membrane. The delivery of molecules, like DNA, peptides, siRNA, proteins and impermeable inhibitors to universal cell lines, including challenging neurons and immune cells, has been demonstrated with this method. A recent study suggests that puncture does not occur upon initial contact between the cell and nanoneedle, but due to the active forces generated by cell spreading and formation of tension-promoting focal adhesions . Certain criteria must be met by the nanoneedles, such as aspect ratio, pitch, sharpness and Young’s Modulus, in order to achieve good cell membrane penetration [54, 55]. Vertical silicon nanowires being used as a universal platform for delivering biomolecules into living cells; [56, 57] these vertical silicon nanowire arrays can be fabricated by different means, such as template-assisted dry etching, metal-assisted silicon etching or conventional top-down lithography. Scalable nanofabrication techniques such as nanosphere lithography can be readily used to generate nanoneedles with sub-50 nm diameters or hierarchical nanotubes. The dimensions and pitches of the silicon nanowires are also tunable to generate different levels of force on the cells. This variable application of forces has also been used to manipulate the growth of stem cells [58, 59].
Active puncture and passive delivery strategy
As shown in Fig. 3b, biomolecular cargos diffuse from the extracellular medium through the transient nanopores after withdrawal of the needles. It is worth noting that this mode is quite similar to other physical approaches such as electroporation and cell squeeze, which also generate transient nanopores on the cell surface. The cellular membrane recovers within a short period of time. In this method, a standard laboratory centrifuge was used to spin down a nanoneedle array substrate facing the adherent cultured cells, followed by withdrawal of the array and diffusive entry of cargo from the medium. A wide variety of cargos including DNA plasmids, RNA and proteins have been demonstrated to be successfully delivered to different cell types while maintaining > 80% viability [53, 60]. Biodegradable porous silicon needles were also developed to deliver different types of cargos. Since the active puncture is needed to facilitate entry of the molecules of interest, the required force for effective membrane penetration was investigated and estimated to be 2 nN per needle for needles with diameter of ~ 300 nm and height of ~ 4 μm . Reduction in the needle diameter has been shown to help reduce the needed penetration force, thus reducing the required centrifuge speed.
Passive puncture and active delivery strategy
In Fig. 3c, hollow nanoneedles (also called nanotubes or nanostraws) are used to enable direct injection of target molecules (ionic species, DNA plasmids) into the cells after cellular membrane penetration with the nanostructures [17, 61, 62]. However, the passive puncture mode is still involved in this system and thus, a set of suitable nanotube mechanical parameters are needed for effective membrane penetration. The nanotubes or nanostraws can be fabricated using a porous membrane (such as porous polycarbonate) as the template . These nanostraws allow for direct intracellular access without perturbing vital cell functions. A key benefit of this configuration is real-time control over delivery dynamics, volume and dosage concentration, as well as possible gating with electric fields.
Summary and perspective
In summary, the recent progresses on the precisely engineered 1D microstructures have shown great potential in revolutionizing both of the conventional intradermal and intracellular delivery. The microneedle patch enabled intradermal delivery brought unprecedent convenience to the patients as well as doctors with even improved efficiency in the drug deliver and release. Due to its simplicity, microneedle patch is expected to help improve the vaccine coverage in developing countries . In addition, smart micropatch systems are also being developed to for the health monitoring and disease diagnose. The nanoneedle arrays are playing a critical role in the development of next-generation intracellular delivery technologies to greatly facilitate progress in multiple fields from cell-based therapies. They take us beyond routine nucleic acid transfection and enable robust manipulation of previously recalcitrant cell types. Including nanoneedles, other intracellular platforms based on exploding bubbles , microfluidic squeezing [66, 67] have been transformed into commercial ventures. Tackling this up and coming age of issues may depend on our capacity to comprehend current conveyance components and to execute the scientific methodologies important to describe cell reactions. Notwithstanding the hindrances that remain, we foresee that cutting edge innovations will make an interpretation of past scholastic undertakings into versatile, customized, cell-based diagnostics and the utilization of clinical intracellular conveyance to design cell destiny for helpful advantage.
The authors acknowledge the helpful discussions with Dr. Steven J. Jonas and Ms. Isaura M. Frost from University of California Los Angeles.
X.X. and W.J. acknowledges the support from Tongji University. L.M. acknowledges the support from National Natural Science Foundation of China under Grants 51875518, 81501607 and 51475419, Key Research and Development Projects of Zhejiang Province under Grant 2017C01054.
Compliance with ethical standards
Conflict of interest
WJ, L.M and X.X declare that they have no conflict of interest.
This review does not contain any studies with human or animal subjects performed by any of the authors.
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