Microneedle Insertion Research Articles

Hollow Microneedle for Drug Delivery Applications to Improve Patient Compliance

In recent years, microfluidic Drug Delivery System (DDS) using microneedle have been given much attention as a novel way for transdermal drug delivery. In this paper, the orthogonal array design is applied for the first time to optimize the bevel tip hollow microneedle suitable for efficient transdermal drug delivery applications. An analytical model has been presented considering the puncture force and friction forces acting on the microneedle during insertion into the soft tissue. The stiffness coefficients are derived as a function of the internal behaviour of the microneedle. Based on these outcomes, an L16 orthogonal Taguchi experiment with two factors, each having four levels, has been utilized to determine the optimal microneedle tip angle (α0) and insertion force (F) for successful penetration of microneedle into the soft tissue during drug delivery. The output responses accounted for investigation are the stiffness coefficients, forces and moments acting on the microneedle, deflection, strain and contact stress. These output response variables must be optimal to avoid failure of microneedle and their effects are studied using Analysis of Variance (ANOVA) to determine the relationship between the levels and the responses. The Taguchi experiment performed using Signal-to-Noise Ratio suggests that microneedle tip angle of 10o and insertion force of 0.25N is optimal. The microneedle tip angle is found to be the most significant control factor for the robust design of hollow microneedle to obtain successful insertion of microneedle into the soft tissue during drug delivery applications.

Methylene Blue-Loaded Dissolving Microneedles: Potential Use in Photodynamic Antimicrobial Chemotherapy of Infected Wounds.

Photodynamic therapy involves delivery of a photosensitising drug that is activated by light of a specific wavelength, resulting in generation of highly reactive radicals. This activated species can cause destruction of targeted cells. Application of this process for treatment of microbial infections has been termed “photodynamic antimicrobial chemotherapy” (PACT). In the treatment of chronic wounds, the delivery of photosensitising agents is often impeded by the presence of a thick hyperkeratotic/necrotic tissue layer, reducing their therapeutic efficacy. Microneedles (MNs) are an emerging drug delivery technology that have been demonstrated to successfully penetrate the outer layers of the skin, whilst minimising damage to skin barrier function. Delivering photosensitising drugs using this platform has been demonstrated to have several advantages over conventional photodynamic therapy, such as, painless application, reduced erythema, enhanced cosmetic results and improved intradermal delivery. The aim of this study was to physically characterise dissolving MNs loaded with the photosensitising agent, methylene blue and assess their photodynamic antimicrobial activity. Dissolving MNs were fabricated from aqueous blends of Gantrez® AN-139 co-polymer containing varying loadings of methylene blue. A height reduction of 29.8% was observed for MNs prepared from blends containing 0.5% w/w methylene blue following application of a total force of 70.56 N/array. A previously validated insertion test was used to assess the effect of drug loading on MN insertion into a wound model. Staphylococcus aureus, Escherichia coli and Candida albicans biofilms were incubated with various methylene blue concentrations within the range delivered by MNs in vitro (0.1–2.5 mg/mL) and either irradiated at 635 nm using a Paterson Lamp or subjected to a dark period. Microbial susceptibility to PACT was determined by assessing the total viable count. Kill rates of >96%, were achieved for S. aureus and >99% for E. coli and C. albicans with the combination of PACT and methylene blue concentrations between 0.1 and 2.5 mg/mL. A reduction in the colony count was also observed when incorporating the photosensitiser without irradiation, this reduction was more notable in S. aureus and E. coli strains than in C. albicans.

The Troy Microneedle: A Rapidly Separating, Dissolving Microneedle Formed by Cyclic Contact and Drying on the Pillar (CCDP).

In dissolving microneedle (DMN)-mediated therapy, complete and rapid delivery of DMNs is critical for the desired efficacy. Traditional patch-based DMN delivery, however, may fail due to incomplete delivery from insufficient skin insertion or rapid separation of microneedles due to their strong bond to the backing film. Here, we introduce the Troy microneedle, which was created by cyclic contact and drying on the pillar (CCDP), and which enabled simultaneous complete and rapid delivery of DMN. This CCDP process could be flexibly repeated to achieve a specific desired drug dose in a DMN. We evaluated DMN separation using agarose gel, and the Troy microneedle achieved more complete and rapid separation than other, more deeply dipped DMN, primarily because of the Troy’s minimal junction between the DMN and pillar. When Troy microneedles were applied to pig cadaver skin, it took only 15 s for over 90% of encapsulated rhodamine B to be delivered, compared to 2 h with application of a traditional DMN patch. In vivo skin penetration studies demonstrated rapid DMN-separation of Troy microneedles still in solid form before dissolution. The Troy microneedle overcomes critical issues associated with the low penetration efficiency of flat patch-based DMN and provides an innovative route for DMN-mediated therapy, combining patient convenience with the desire drug efficacy.

Analytical approach for optimization design of MEMS based microneedles in drug delivery system

Microneedles have been recently given much attention as a novel way for transdermal drug delivery. In this paper, the mechanics of microneedle insertion into soft tissue and also the changes in the forces and moments of the microneedle is to be presented. A non-linear displacement of the microneedle insertion into the soft tissue is expressed as a quadratic polynomial function and solved using Galerkin technique. The geometrical behavior of the microneedle tip is to be considered in the analytical model. The microneedle insertion is classified into two phases namely, pre-puncture and post-puncture. An analytical model of microneedle stiffness coefficient and effect of interfacial contact force in frictionless condition are derived using Love’s thin rod theory. The interfacial contact stresses at the interface between the microneedle and soft tissue are obtained from the contact forces using the Hertzian theory. Statistical analysis based on the analytical model was done for studying various effects of process variables on the dependent variables. The statistical analysis was done by Analysis of variance (ANOVA). The graphical analysis of the analytical model are presented using MATLAB. The analytical model helps to improve the microneedle design by minimizing the pain of insertion and increasing the mechanical strength.

Poly-γ-glutamic acid microneedles with a supporting structure design as a potential tool for transdermal delivery of insulin

Incomplete insertion is a common problem associated with polymer microneedles (MNs) that results in a limited drug delivery efficiency and wastage of valuable medication. This paper presents a fully insertable MN system that is composed of poly-γ-glutamic acid (γ-PGA) MNs and polyvinyl alcohol (PVA)/polyvinyl pyrrolidone (PVP) supporting structures. The PVA/PVP supporting structures were designed to provide an extended length for counteracting skin deformation during insertion and mechanical strength for fully inserting the MNs into the skin. When inserted into the skin, both the supporting structures and MNs can be dissolved in the skin within 4min, thus quickly releasing the entire drug load from the MNs. To evaluate the feasibility and reproducibility of using the proposed system for treating diabetes, we administered insulin-loaded MNs to diabetic rats once daily for 2days. The results indicated that the hypoglycemic effect in the rats receiving insulin-loaded MNs was comparable to that observed in rats receiving subcutaneous insulin injections. The relative pharmacological availability and relative bioavailability of the insulin were in the range of 90–97%, indicating that the released insulin retained its pharmacological activity. We observed no significant differences in the plasma insulin concentration profiles between the first and second administrations, confirming the stability and accuracy of using the proposed MN system for insulin delivery. These results indicated that the γ-PGA MNs containing the supporting structure design enable complete and efficient delivery of encapsulated bioactive molecules and have great potential for the relatively rapid and convenient transdermal delivery of protein drugs. Statement of SignificanceIncomplete insertion of microneedles largely limits drug delivery efficiency and wastage of valuable medication. To address this problem, we developed a fully insertable poly-glutamic acid microneedles with a supporting structure design to ensure complete and efficient delivery of encapsulated drugs. The supporting structures were designed to provide an extended length for counteracting skin compressive deformation during puncture and mechanical strength for fully inserting the microneedles into the skin. When inserted into the skin, both the supporting structures and microneedles can be dissolved in the skin within 4min, thus quickly releasing the entire drug load. This study demonstrated that the proposed microneedle system featuring this unique design allows more convenient and efficient self-administration of drugs into the skin.

Enhanced skin delivery of vismodegib by microneedle treatment.

The present study investigated the effects of microneedle treatment (maltose microneedles, Admin Pen™ 1200, and Admin Pen™ 1500) on in vitro transdermal delivery of vismodegib with different needle lengths, skin equilibration times, and microneedle insertion durations. The influence of microneedle treatment on the dimensions of microchannels (dye binding, calcein imaging, histology, and confocal microscopy studies), transepidermal water loss, and skin permeability of vismodegib was also evaluated. Skin viscoelasticity was assessed using a rheometer, and microneedle geometry was characterized by scanning electron microscopy. Permeation studies of vismodegib through dermatomed porcine ear skin were conducted using vertical Franz diffusion cells. Skin irritation potential of vismodegib formulation was assessed using an in vitro reconstructed human epidermis model. Results of the in vitro permeation studies revealed significant enhancement in permeation of vismodegib through microneedle-treated skin. As the needle length increased from 500 to 1100 and 1400μm, drug delivery increased from 14.50 ± 2.35 to 32.38 ± 3.33 and 74.40 ± 15.86μg/cm(2), respectively. Positive correlation between drug permeability and microneedle treatment duration was observed. The equilibration time was also found to affect the delivery of vismodegib. Thus, changes in microneedle length, equilibration time, and duration of treatment altered transdermal delivery of vismodegib.

Review of patents for microneedle application devices allowing fluid injections through the skin.

Microneedles have been developed in the past few years as a new means of transdermal drug delivery. They indeed present many advantages compared to injections using hypodermic needles (reduced risk of contamination and epidermic reactions), but mostly bring comfort and compliance to patients. Microneedles may be plain, opening pathways for medications to dissolve into the skin, or hollow, allowing fluid to actually enter the dermis or the hypodermis. This review focuses on the latter type of microneedles and two issues with their application: first, ensuring correct insertion into the skin (controlled and repeatable insertion depth, mainly); and second, ensuring correct fluid delivery to the dermis (controlled infusion rate). This paper thus focuses on recently published patents for hollow microneedle applicators-i.e., microneedles applicators that allow fluid delivery to the skin. Descriptions are given of several of the most relevant patents concerning this. The benefits and drawbacks of the different solutions are also described.

Microneedle characterisation: the need for universal acceptance criteria and GMP specifications when moving towards commercialisation.

With interest in microneedles as a novel drug transdermal delivery system increasing rapidly since the late 1990s (Margetts and Sawyer Contin Educ Anaesthesia Crit Care Pain. 7(5):171-76, 2007), a diverse range of microneedle systems have been fabricated with varying designs and dimensions. However, there are still very few commercially available microneedle products. One major issue regarding microneedle manufacture on an industrial scale is the lack of specific quality standards for this novel dosage form in the context of Good Manufacturing Practice (GMP). A range of mechanical characterisation tests and microneedle insertion analysis techniques are used by researchers working on microneedle systems to assess the safety and performance profiles of their various designs. The lack of standardised tests and equipment used to demonstrate microneedle mechanical properties and insertion capability makes it difficult to directly compare the in use performance of candidate systems. This review highlights the mechanical tests and insertion analytical techniques used by various groups to characterise microneedles. This in turn exposes the urgent need for consistency across the range of microneedle systems in order to promote innovation and the successful commercialisation of microneedle products.

Impact insertion of transfer-molded microneedle for localized and minimally invasive ocular drug delivery

It has been challenging for microneedles to deliver drugs effectively to thin tissues with little background support such as the cornea. Herein, we designed a microneedle pen system, a single microneedle with a spring-loaded microneedle applicator to provide impact insertion. To firmly attach solid microneedles with 140μm in height at the end of macro-scale applicators, a transfer molding process was employed. The fabricated microneedle pens were then applied to mouse corneas. The microneedle pens successfully delivered rhodamine dye deep enough to reach the stromal layer of the cornea with small entry only about 1000μm2. When compared with syringes or 30G needle tips, microneedle pens could achieve more localized and minimally invasive delivery without any chances of perforation. To investigate the efficacy of microneedle pens as a way of drug delivery, sunitinib malate proven to inhibit in vitro angiogenesis, was delivered to suture-induced angiogenesis model. When compared with delivery by a 30G needle tip dipped with sunitinib malate, only delivery by microneedle pens could effectively inhibit corneal neovascularization in vivo. Microneedle pens could effectively deliver drugs to thin tissues without impairing merits of using microneedles: localized and minimally invasive delivery.

Lidocaine permeation from a lidocaine NaCMC/gel microgel formulation in microneedle-pierced skin: vertical (depth averaged) and horizontal permeation profiles.

Common local anaesthetics such as lidocaine are administered by the hypodermic parenteral route but it causes pain or anxiety to patients. Alternatively, an ointment formulation may be applied which involves a slow drug diffusion process. In addressing these two issues, this paper aims to understand the significance of the 'poke and patch' microneedle (MN) treatment on skin in conjunction to the lidocaine permeation, and in particular, the vertical (depth averaged) and horizontal (e.g. lateral) permeation profiles of the drug in the skin. The instantaneous pharmacokinetics of lidocaine in skin was determined by a skin denaturation technique coupled with Franz diffusion cell measurements of the drug pharmacokinetics. All pharmacokinetic profiles were performed periodically on porcine skin. Three MN insertion forces of 3.9, 7.9 and 15.7N were applied on the MN to pierce the skin. For the smaller force (3.9N), post MN-treated skin seems to provide an 'optimum' percutaneous delivery rate. A 10.2-fold increase in lidocaine permeation was observed for a MN insertion force of 3.9N at 0.25h and similarly, a 5.4-fold increase in permeation occurred at 0.5h compared to passive diffusional delivery. It is shown that lidocaine permeates horizontally beyond the area of the MN-treated skin for the smaller MN insertion forces, namely, 3.9 and 7.9N from 0.25 to 0.75h, respectively. The lateral diffusion/permeation of lidocaine for larger MN-treated force (namely, 15.7N in this work) seems to be insignificant at all recorded timings. The MN insertion force of 15.7N resulted in lidocaine concentrations slightly greater than control (passive diffusion) but significantly less than 3.9 and 7.9N impact force treatments on skin. We believe this likelihood is due to the skin compression effect that inhibits diffusion until the skin had time to relax at which point lidocaine levels increase.

Feasibility study for intraepidermal delivery of proteins using a solid microneedle array

Solid microneedles (MN) are a promising tool for dermal drug delivery. Particular focus lies on the field of vaccination due to pain-free, safe, hygienic and patient compliant antigen deposition. Diverse coating techniques and formulations have been developed to preserve vaccine activity and to enable targeted drug deposition in the skin. Process and long-term storage stability of coated MN, however, have not yet been studied in detail. Hence, a feasibility study was conducted determining the appropriate needle length (300μm) for local intraepidermal protein delivery. Moreover, a protein-stabilizing coating formulation was developed. Coating of the MN resulted in protein concentrations between 10 and 23μg, 90% of the bioactivity of the model protein asparaginase was maintained for 3 months. Skin experiments verified the intraepidermal deposition of 68.0±11.7% of the coated model protein after single application. Slightly increased interleukin 8 levels right after MN insertion indicated minor skin irritation due to the mechanical piercing stress. Thus, specifically highlighting protein stabilization during storage, we demonstrated that selective intraepidermal deposition of proteins or peptides’ using solid MN is a feasible approach.

Hollow polymer microneedles array resistance and insertion tests

Microneedles are developed in order to become the transdermal administration method of the future. They however still face numerous challenges. This paper addresses the challenge to effectively insert the microneedle arrays into membranes. A recently proposed model membrane and test method for microneedles insertion, published in International Journal of Pharmaceutics, is used in this aim. A moulded 4 by 4 hollow polymer microneedle array developed at the Université Libre de Bruxelles is tested for insertion using this model. Results show that the array is extremely resistant to insertion, it can withstand very high forces and even multiple insertions without blunting. Different insertion tests were performed on a folded in eight Parafilm® film because it exhibits excellent similarity to porcine skin. The insertion force, the insertion speed and the holding time of the array against membranes must be optimised in order to get efficient reliable insertions at, at least, 500μm depth.

Perivascular biodegradable microneedle cuff for reduction of neointima formation after vascular injury.

Restenosis often occurs at the site of vascular grafting and may become fatal for patients. Restenosis at anastomosis sites is due to neointimal hyperplasia (NH) and difficult to treat with conventional treatments. Such abnormal growth of smooth muscle cells in tunica media of vascular tissue can be reduced by delivering anti-proliferation drugs such as paclitaxel (PTX) to the inner vascular layer. Drug eluting stents (DES) or drug eluting balloon (DEB) have been developed to treat such vascular diseases. However, they are less efficient in drug delivery due to the drug loss to blood stream and inadequate to be applied to re-stenotic area in the presence of stent or anastomosis sites. Recently, we have introduced microneedle cuff (MNC) as perivascular delivery devices to achieve high delivery efficiency to tunica media. In this study, we investigated in vivo microneedle insertion and efficacy in treating NH using a rabbit balloon injury model. Microneedle shape was optimized for reliable insertion into tunica media layer. Uniform distribution of PTX in tunica media delivered by MNC devices was also confirmed. Animal study demonstrated significant NH reduction by MNC treatments and much higher delivery efficiency than flat type devices.

Optimization of Impedance Spectroscopy Techniques for Measuring Cutaneous Micropore Formation after Microneedle Treatment in an Elderly Population

The objective of this study was to optimize a reproducible impedance spectroscopy method in elderly subjects as a means to evaluate the effects of microneedles on aging skin. Human volunteers were treated with microneedles at six sites on the upper arm. Repeated impedance measurements were taken pre- and post-microneedle insertion. Two electrode types were evaluated (dry vs. gel), using either light or direct pressure to maintain contact between the electrode and skin surface. Transepidermal water loss (TEWL) was measured as a complementary technique. Five control subjects and nine elderly subjects completed the study. Microneedle insertion produced a significant decrease in impedance from baseline in all subjects (p < 0.05, regardless of electrode type or pressure application), confirming micropore formation. This was supported by a complementary significant increase in TEWL (p < 0.05). The gel*direct condition produced the lowest variability between measurements, as demonstrated by a coefficient of variation of 3.8% and 3.5% (control and elderly subjects, respectively). This was lower than variation between TEWL measurements at the same sites: 19.8% and 21.6% (control and elderly subjects, respectively). Impedance spectroscopy reproducibly measures micropore formation in elderly subjects, which will be essential for future studies describing microneedle-assisted transdermal delivery in aging populations.

A proposed model membrane and test method for microneedle insertion studies

A commercial polymeric film (Parafilm M®, a blend of a hydrocarbon wax and a polyolefin) was evaluated as a model membrane for microneedle (MN) insertion studies. Polymeric MN arrays were inserted into Parafilm M® (PF) and also into excised neonatal porcine skin. Parafilm M® was folded before the insertions to closely approximate thickness of the excised skin. Insertion depths were evaluated using optical coherence tomography (OCT) using either a force applied by a Texture Analyser or by a group of human volunteers. The obtained insertion depths were, in general, slightly lower, especially for higher forces, for PF than for skin. However, this difference was not a large, being less than the 10% of the needle length. Therefore, all these data indicate that this model membrane could be a good alternative to biological tissue for MN insertion studies. As an alternative method to OCT, light microscopy was used to evaluate the insertion depths of MN in the model membrane. This provided a rapid, simple method to compare different MN formulations. The use of Parafilm M®, in conjunction with a standardised force/time profile applied by a Texture Analyser, could provide the basis for a rapid MN quality control test suitable for in-process use. It could also be used as a comparative test of insertion efficiency between candidate MN formulations.

Impact-insertion applicator improves reliability of skin penetration by solid microneedle arrays.

Microneedles are needle-like structures shorter than 1 mm that have been advocated as devices for enabling potentially pain-free intradermal delivery of biomacromolecules (1–5). To permit a reproducible delivery of the drug, microneedles should be inserted into the skin in a controlled and reproducible manner (4,6). Obviously, one of the factors that influence the penetration ability of microneedles is the insertion process itself, e.g., microneedles can be inserted manually or by using insertion devices (3,4,6–9). Recently, the penetration ability of low-density arrays (42 microneedles/cm2) with 750-μm long microneedles, applied manually or by using a snap-based applicator, was investigated (10). However, no studies have been reported on the efficiency and reproducibility of the insertion of high-density microneedles into human skin. Therefore, the aim of this study was to investigate the effect of the type of application on inter and intra individual variability of microneedle insertion into human skin by microneedle users. We show that participants using an impact-insertion applicator inserted high-density microneedles into ex vivo human skin with high efficiency and with a low inter and intra individual variation. However, when the same microneedle arrays were inserted by using a manual insertion device, the penetration efficiency was reduced by approximately 40% with a considerably lower reproducibility. Finally, the applicability of an impact-insertion applicator/microneedle array was confirmed in a vaccination study in mice.

Vaccine Delivery to the Oral Cavity Using Coated Microneedles Induces Systemic and Mucosal Immunity

The objective of this study is to evaluate the feasibility of using coated microneedles to deliver vaccines into the oral cavity to induce systemic and mucosal immune responses. Microneedles were coated with sulforhodamine, ovalbumin and two HIV antigens. Coated microneedles were inserted into the inner lower lip and dorsal surface of the tongue of rabbits. Histology was used to confirm microneedle insertion, and systemic and mucosal immune responses were characterized by measuring antigen-specific immunoglobulin G (IgG) in serum and immunoglobulin A (IgA) in saliva, respectively. Histological evaluation of tissues shows that coated microneedles can penetrate the lip and tongue to deliver coatings. Using ovalbumin as a model antigen it was found that the lip and the tongue are equally immunogenic sites for vaccination. Importantly, both sites also induced a significant (p < 0.05) secretory IgA in saliva compared to pre-immune saliva. Microneedle-based oral cavity vaccination was also compared to the intramuscular route using two HIV antigens, a virus-like particle and a DNA vaccine. Microneedle-based delivery to the oral cavity and the intramuscular route exhibited similar (p > 0.05) yet significant (p < 0.05) levels of antigen-specific IgG in serum. However, only the microneedle-based oral cavity vaccination group stimulated a significantly higher (p < 0.05) antigen-specific IgA response in saliva, but not intramuscular injection. In conclusion, this study provides a novel method using microneedles to induce systemic IgG and secretory IgA in saliva, and could offer a versatile technique for oral mucosal vaccination.

Design of MEMS Based Microneedle for Drug Delivery System

Microneedles have been recently given much attention as a novel way for transdermal drug delivery. In this paper, the mechanics of microneedle insertion into soft tissue during drug delivery and also the changes in the forces and moments of the microneedle is to be presented. A non-linear displacement of the microneedle insertion into the soft tissue is expressed as a quadratic polynomial function and solved using Galerkin technique. The geometrical behavior of the microneedle tip is to be considered in the analytical model. The graphical analysis of the analytical model are presented using MATLAB. The analytical model helps to improve the microneedle design by minimizing the pain of insertion and increasing the mechanical strength.

The Deformation and Fracture of Skin under Insertion of Microneedle

Microneedle, as a novel way for transdermal drug delivery, due to its painless and minimally invasive, has recently attracted much attention. The present study investigated skin deformation and failure due to insertion by a single microneedle with nonlinear finite element method. The finite element model considered multilayered, non-linear and hyperelastic properties of the skin, and tissue failure was phenomenologically described by an effective stress failure criterion. The deformation and failure of skin and the force displacement behaviour of the microneedle are discussed. The numerical results show a good agreement with the reported experimental data. The performed study provided novel insights into skin failure due to insertion of microneedle, and provided useful information with which to optimise the microneedle design.

Microprecision Delivery of Oligonucleotides in a 3D Tissue Model and Its Characterization Using Optical Imaging

Despite significant potential of oligonucleotides (ONs) for therapeutic and diagnostic applications, rapid and widespread intracellular delivery of ONs in cells situated in tissues such as skin, head and neck cavity, and eye has not been achieved. This study was aimed at evaluating the synergistic combination of microneedle (MN) arrays and biochemical approaches for localized intratissue delivery of oligonucleotides in living cells in 3D tissue models. This synergistic combination was based on the ability of MNs to precisely deliver ONs into tissues to achieve widespread distribution, and the ability of biochemical agents (streptolysin O (SLO) and cholesterol conjugation to ONs) to enhance intracellular ON delivery. The results of this study demonstrate that ON probes were uniformly coated on microneedle arrays and were efficiently released from the microneedle surface upon insertion in tissue phantoms. Co-insertion of microneedles coated with ONs and SLO into 3D tissue models resulted in delivery of ONs into both the cytoplasm and nucleus of cells. Within a short incubation time (35 min), ONs were observed both laterally and along the depth of a 3D tissue up to a distance of 500 μm from the microneedle insertion point. Similar widespread intratissue distribution of ONs was achieved upon delivery of ON-cholesterol conjugates. Uniformity of ON delivery in tissues improved with longer incubation times (24 h) postinsertion. Using cholesterol-conjugated ONs, delivery of ON probes was limited to the cytoplasm of cells within a tissue. Finally, delivery of cholesterol-conjugated anti-GFP ON resulted in reduction of GFP expression in HeLa cells. In summary, the results of this study provide a novel approach for efficient intracellular delivery of ONs in tissues.