biodegradablesiliconnanoneedlesdelivering...

ARTICLESPUBLISHED ONLINE: 30 MARCH 2015 | DOI: 10.1038/NMAT4249

Biodegradable silicon nanoneedles deliveringnucleic acids intracellularly induce localizedin vivo neovascularizationC. Chiappini1,2†, E. De Rosa3†, J. O. Martinez3, X. Liu3, J. Steele1,2, M. M. Stevens1,2*and E. Tasciotti3*

The controlled delivery of nucleic acids to selected tissues remains an ine�cient process mired by low transfection e�cacy,poor scalability because of varying e�ciency with cell type and location, and questionable safety as a result of toxicity issuesarising from the typical materials and procedures employed. High e�ciency and minimal toxicity in vitro has been shownfor intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery hasbeen di�cult, as current interfacing strategies rely on complex equipment or active cell internalization through prolongedinterfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etchingof silicon can access the cytosol to co-deliver DNA and siRNA with an e�ciency greater than 90%, and that in vivo thenanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in bloodperfusion in a target region of the muscle.

The e�cacy of protein and peptide therapy on biologicalprocesses1,2 and the ability of nucleic acid to modulate geneexpression3 has fuelled a renewed interest in the delivery of

biological therapeutics for the study of tissue function. In particular,the localized transfer of genetic material to temporarily alter tissuestructure and function in a desired spatial pattern may enable thestudy of biologically initiated processes in the context of live adulttissues4. Nucleic acids act on intracellular targets and must localizein an active form to either the cytosol or the nucleus to be e�ective.Being large charged molecules, they cannot easily cross the plasmamembrane unless actively ferried5. Ine�cient transfection5–7, safetyconcerns8,9, limited site accessibility10 and poor scalability11 stillhamper the in vivo delivery of nucleic acids. Nanoneedles cane�ciently transfer bioactive agents to many cells in parallel12, sensetheir electrical activity13, or transduce optical information fromwithin the cytoplasm14 with minimal impact on viability15 andmetabolism16. The mechanism for nanoneedle-mediated cytosolicdelivery is still highly debated; conflicting evidence supports eithercytosolic display of the nanoneedles12,17 or cell permeabilization duetomembrane deformation18,19. Nanoneedles at present rely on activecell processes17,20 or complex equipment for payload delivery21,22,limiting their use in vivo.

We hypothesized that in vivo patterning of gene expressioncould be mediated by the intracellular delivery of nucleic acidsthrough their injection by vertical arrays of biodegradableporous nanoneedles.

Characterization of porous silicon nanoneedlesPorous silicon is a biodegradable23,24 material amenable tomicrofabrication25, with favourable toxicology and versatile deliveryproperties26,27 for systemic27,28 and intra-organ29 administrationas well as for the manufacturing of implantable devices30. The

nanoneedles were fabricated by combining metal-assisted chemicaletch31 with standard microfabrication (Fig. 1a–c). Their geometrywas tailored to optimize the fundamental parameters necessaryfor intracellular delivery, including mechanical stability, cell-membrane penetration, cytotoxicity and loading capacity (porevolume). The tunable tip diameter was reduced to less than 50 nm,amply below the cuto� diameter established for nanoneedlecytotoxicity32 (Fig. 1c). The prototypical porous conical nanoneedlehad 5 µm length, 50 nm apical width and 600 nm base diameter(Fig. 1b), providing an over 300-fold increase in surface area forpayload adsorption compared to a solid cylindrical nanowireof equivalent apical diameter (2.4 ⇥ 108 versus 7.8 ⇥ 105 nm2).The needles’ porosity could be tailored between 45% and 70%,allowing the modulation of degradation time, payload volumeand mechanical properties31. Conical nanoneedles were extremelyrobust, as confirmed by their structural integrity following in vitroand in vivo applications (Supplementary Fig. 1). A failure load of260 nN and a critical buckling load of 264 nN were estimated forthe nanoneedles; two orders of magnitude higher than the forcesexperienced during active penetration of a cell (0.5 to 2 nN; ref. 32;Supplementary Information). Compression testing highlightedthe onset of nanoneedle failure at 3.1 µN, with terminal failureoccurring at 6.2 µN (Supplementary Fig. 2).

Nanoneedles progressively dissolved over time in physiologicconditions (Fig. 1d,e), first thinning and increasing in porosity,and ultimately losing their shape after 15 h. By 72 h only theirsolid stump remained. The atomic quantification of Si released insolution confirmed the progressive degradation of the nanoneedles,which was substantially achieved within 36 h (Fig. 1e). This uniquedegradation behaviour, not exhibited by solid nanoneedles, enablesour system to temporarily interface with cells, and to completelydissolve over time.

1Department of Materials, Imperial College London, London SW6 7PB, UK. 2Department of Bioengineering and Institute of Biomedical Engineering,Imperial College London, London SW6 7PB, UK. 3Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas 77030, USA.†These authors contributed equally to this work. *e-mail: [email protected]; [email protected]

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NATUREMATERIALS DOI: 10.1038/NMAT4249 ARTICLES

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Figure 1 | Porous silicon nanoneedles. a, Schematic of the nanoneedle synthesis combining conventional microfabrication and metal-assisted chemicaletch (MACE). RIE, Reactive ion etching. b,c, SEM micrographs showing the morphology of porous silicon nanoneedles fabricated according to the processoutlined in a. b, Ordered nanoneedle arrays with pitches of 2 µm, 10 µm and 20 µm, respectively. Scale bars, 2 µm. c, High-resolution SEM micrographs ofnanoneedle tips showing the nanoneedles’ porous structure and the tunability of tip diameter from less than 100 nm to over 400 nm. Scale bars, 200 nm.d, Time course of nanoneedles incubated in cell-culture medium at 37 �C. Progressive biodegradation of the needles appears, with loss of structuralintegrity between 8 and 15 h. Complete degradation occurs at 72 h. Scale bars, 2 µm. e, ICP-AES quantification of Si released in solution. Blue and black barsrepresent the rate of silicon release per hour and the cumulative release of silicon, respectively, at each timepoint, expressed as a percentage of total siliconreleased. Error bars represent the s.d. of 3–6 replicates.

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NATURE MATERIALS | VOL 14 | MAY 2015 | www.nature.com/naturematerials 533




ARTICLES NATUREMATERIALS DOI: 10.1038/NMAT4249

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Figure 3 | Intracellular co-delivery of nucleic acids. a, Co-delivery of GAPDH-siRNA (Cy3 labelled in yellow) and GFP plasmid (GFP in green) to cells inculture after application using the nN-B interfacing strategy. Confocal image acquired 48 h post transfection. The siRNA and GFP signal are presentdi�usely throughout the cytosol. Scale bars, 5 µm. b, Flow-cytometry scatter (dot) plot shows that >90% of siRNA transfected cells are positive for Cy3(blue cluster) and that greater than 90% of co-transfected cells are positive for GFP and Cy3 (red cluster) compared with cells transfected with emptyneedles (black). c, Quantification of flow-cytometry data according to the gate outlined in b to show the percentage of GAPDH-positive, GFP-positive anddouble-positive cells for nanoneedles loaded with siRNA (S), siRNA and GFP plasmid (S+P). Empty needles were used as the control (C). d, In-cell westernassay showing an entire nN-B chip seeded with cells. The fluorescent signal from GAPDH is significantly reduced for cells to which GAPDH siRNA has beendelivered (NN-G) relative to empty nanoneedles (NN), nanoneedles loaded with scrambled siRNA (NN-Sc), and empty nanoneedles with GAPDH siRNAdelivered from solution (NN+sG). e, Quantification of the in-cell western assay, showing statistically significant silencing of GAPDH expression to <20%of basal level only in cells treated with nanoneedles loaded with GAPDH. Basal level was evaluated for cells grown on empty nanoneedles. ⇤⇤⇤p<0.001.Error bars represent the s.d. of 3 replicates.

Gene regulation by nanoinjection of nucleic acidsCells were interfaced with nanoneedles (that is, nanoinjected) eitherby seeding themover the nanoneedles (needles on bottom, nN-B) orby pressing the nanoneedles over a monolayer of cells (needles ontop, nN-T). Although the nN-B setting was crucial to understandthe cell–nanoneedle interface, nN-T better mimicked the in vivodelivery setting. When cells were seeded over the nanoneedles, theyappeared to interface with the cell membrane within 4 h (Fig. 2a),whereas the nN-T strategy yielded immediate interfacing (Fig. 2b).Under either condition the cells retained physiologic metabolicactivity and normal proliferation over the course of five days(Fig. 2c). Furthermore, nanoinjection did not induce significanttoxicity. According to the lactate dehydrogenase (LDH) assay, theconcentration of the enzyme in the culturemediumwas unaltered bythe needles, suggesting that nanoinjection did not induce the leakageof intracellular material (Fig. 2d).

Nanoneedles e�ciently loaded and retained nucleic acids,releasing them over 12–18 h (Supplementary Figs 3 and 4). Bothnanoinjection strategies delivered nucleic acids intracellularly andwere able to regulate gene expression. After 30min of nN-Bnanoinjection, siRNA delivery to cells was minimal. However, at24 h and 48 h, siRNA was uniformly distributed throughout thecytosol (Fig. 3a and Supplementary Fig. 5). In the nN-T setting,nanoneedles were applied onto cells using a 100 relative centrifugalforce (rcf) acceleration, and left in place for 30min to deliverthe siRNA to the cytosol (Supplementary Fig. 5). Nanoinjectionrequired either cellular activity or application of an external force.When cellular activity was impaired by maintaining the culture at4 �C, nN-T nanoinjection at 0 rcf did not yield any cytosolic deliveryover 4 h (Supplementary Fig. 6). In contrast, forceful nanoinjectionat 4 �C and 100 rcf yielded cytosolic delivery of siRNA.

The co-loading of a Cy3-labelled siRNA and a GFP-expressingDNA plasmid proved the nanoneedles’ ability to co-deliver twotypes of nucleic acids to the same cell (Fig. 3). The transfection ofactive nucleic acids intoHeLa cells was determined by assessingGFPexpression and measuring siRNA fluorescence within nanoinjectedcells (Fig. 3a–c). Transfection e�ciency was greater than 90% at48 h (Fig. 3b,c and Supplementary Fig. 7). Green fluorescence waspresent at 48 h surrounding the stumps of nanoneedles loadedwith Cy3–siRNA (Fig. 3a). This signal could arise from the GFPoriginating either from the cell cytosol33 or from microvesicles insolution34–36, and adsorbed in the mesoporous silicon matrix37. Thee�ect of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)siRNA nanoinjection was confirmed by the 80% (p < 0.001)silencing in GAPDH expression compared to controls and tocells nanoinjected with scrambled siRNA (Fig. 3d,e). No silencingwas observed for nanoinjection with empty needles in thepresence of GAPDH siRNA in solution, supporting the hypothesisof nanoneedle-mediated, direct delivery of the payload in thecell cytoplasm.

Localized nanoinjection to tissueWeoptimized nanoinjection in vivo by applying nanoneedles loadedwith fluorescent dyes to the skin and muscle of test mice. All invivo studies employed nanoneedles with 50-nm-diameter tips anda 2 µm pitch. Eighty per cent of the loaded dye was transferredinto an area of the skin replicating the chip’s size and shape. Theconcentration and localization of the dye was maintained for upto 48 h, even after washing (Fig. 4a,b), and remained close to thesurface (localized patterning; Supplementary Fig. 8). In contrast,topical application or delivery through a flat chip showed a limitedretention and a patchy distribution of the dye across the tissue





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Figure 4 | Nanoneedles mediate in vivo delivery. a, Longitudinal imaging of mice treated with nanoneedles on top of the skin or underneath the skin on theback muscle and loaded with a near-infrared fluorescent dye. The distribution and di�usion of the delivered fluorescent dye was monitored usingwhole-animal fluorescent imaging for 48 h. b, The delivery and di�usion of the dye was measured at several times following 1-min nanoinjection, andplotted to compare delivery kinetics at the di�erent sites of administration. Error bars represent the s.d. of 3 replicates. c, Near-infrared fluorescent imagingon the skin of mice, comparing the delivery of DyLight 800 using a drop (left), flat Si wafer (middle), or nanoneedles (right). d, Intravital confocal image,showing the delivery pattern of dye-loaded nanoneedles. Scale bar, 1 mm.

(Fig. 4c and Supplementary Fig. 9). Unlike microneedles thatshow a gradient of e�ect originating from each puncture site38,nanoinjection provided a more uniform delivery owing to the highdensity of nanoneedles per surface area (Fig. 4d). Nanoinjectionto the ear and to the muscle demonstrated similar uniformity,localization and retention patterns (Fig. 4b and SupplementaryFig. 9). These findings confirmed the ability of the nanoneedlesto confine the treatment to a localized region, regardless of tissuearchitecture, sti�ness and degree of vascularization.

Safety of nanoinjection in vivoTo evaluate in vivo safety, we assessed the impact of nanoinjectionwith empty nanoneedles on di�erent murine tissues at themicro- and nanoscale. Real-time bioluminescent imaging followingadministration of luminol39 showed that nanoinjection did notinduce local acute inflammation in muscle or skin at 5 and 24 h(Fig. 5a,b).

The impact at the site of interfacing was assessed in muscle, skinand ear tissues. Haematoxylin and eosin (H&E) histology and elec-tron microscopy analysis indicated substantial similarity betweenuntreated and nanoinjected tissues. The local myofibres showednormal morphology with minimal disintegration or leukocyte infil-tration (Fig. 5c and Supplementary Fig. 10a). The histological anal-ysis showed intact tissue membranes after nanoinjection. Treatedskin showed preserved epidermis, dermis, and sebaceous-gland andhair-follicle layers typical of nude mice, with no sign of leukocyteinfiltration (Fig. 5c). The skin showed no signs of epidermal, dermalor capillary vessel disruption, with normal keratinocyte levels andminimal thickening of the epidermis as well as negligible signs ofhyperkeratosis or necrotic keratinocytes.

Histological analysis also indicated the impact of nanoinjectionon the overall structure ofmuscle at 5 h, 5 days and 15 days followingtreatment. At each time period the transverse and longitudinalsections revealed myofibres of uniform size and shape analogous toWT muscle. The area, mean diameter and roughness of individualmyofibres were not significantly di�erent fromWT (Fig. 5d,e). Thenuclei were located on the periphery, a feature characteristic of

skeletal muscle cells (Fig. 5c and Supplementary Fig. 10b). H&Ehistology confirmed the absence of infiltrating cells, disintegratingor necrotic myofibres, or scar formation due to the treatment40.TEMhistology ofmuscle revealed a preserved cellular ultrastructure(Fig. 5f,g). The muscles exhibited the conventional sarcomerestructures (A-band, I-band, Z-line and M-line) with transverselyaligned Z-lines within each myofibril. High magnification of themyofibrils, sarcoplasmic reticulum, mitochondria and transversetubules confirmed that the basic units and organelles of the musclesretained a physiologic morphology.

Neovascularization by nanoinjection of VEGF plasmidWe compared the e�ciency of nanoinjection of 100 µg of humanVEGF165 plasmid DNA (pVEGF165) to the direct injection ofthe same amount of naked plasmid into the muscles of mice(Fig. 6). VEGF is a master angiogenic gene, which inducestissue neovascularization, increases vessel interconnectivity,perfusion, local vascular leakiness, and infiltration of circulatingcells41,42. Real-time PCR analysis indicated that both treatmentsinduced expression of human VEGF165 for up to seven days,with nanoinjection exhibiting an average higher expression thandirect injection (Supplementary Fig. 11). In both instances,treated muscles progressively exhibited higher vascularizationcompared with the control over the course of two weeks(Fig. 6a). However, only the neovascularization induced bythe nanoinjection demonstrated a surge in perfusion (Fig. 6b).Also, the neovasculature in nanoinjected muscles exhibited highlyinterconnected and structured vessels in proximity to the surface(Fig. 6a and Supplementary Figs 12–14), and a sixfold increase inoverall blood perfusion (Fig. 6b) and in the number of nodes at14 days (Fig. 6c). On the other hand, the direct injection did notresult in significantly higher perfusion or an increase in numberof nodes (Fig. 6b,c) compared to sham surgery. The moderateincrease in perfusion observed in the control was due to the surgicaltreatment, which required a small incision of the fascia to exposethe muscle. In this case, the increased perfusion was not associatedwith an increase in the number of nodes or with the formation






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Figure 5 | Safety profile of nanoinjection. a, Representative longitudinal bioluminescence images of mice with luminol at 5 and 24 h following nanoneedletreatment to the muscle and skin on the ear. Phorbol-12-myristate-13-acetate (PMA) treatment and surgical incisions were employed as positive controls.b, Quantification of the average radiance exposure acquired from mice administered with luminol at 5 and 24 h for surgical incision (black), PMA-treatedears (green), muscles (blue) and skin of ear (red) following treatment with nanoneedles. Data normalized to control represented by the black dashed line.⇤p<0.05 versus control. Error bars represent the s.d. of 3 replicates. c, H&E and TEM micrographs at the site of nanoinjection for muscle, skin and ear,comparing nanoneedles and control tissues. d, H&E images of muscle sections at 5 h, 5 days and 15 days following nanoinjection compared againstwild-type (WT; that is, control) tissues. e, Quantification of individual myofibres for area, diameter and roughness for the times depicted in d. Error barsrepresent the s.d. of measurements in 34–92 areas (for nN-T: 49, 34 and 92 for 5 h, 5 d and 15 d, respectively; for WT: 60, 90 and 53 for 5 h, 5 d and 15 d,respectively) taken from 3 animals. f,g, TEM micrographs of muscle sections for nanoneedles (f) and control (g) sections, illustrating the impact ofnanoinjection on the ultrastructure of tissues at low magnification. Higher-magnification images to the right depict intact myofibrils (top) andmitochondria (bottom). Red lines, A-band; black lines, I-band; black arrows, M-line; red arrows, Z-line; red crosses, transverse tubules; black stars,sarcoplasmic reticulum; red squares (or red arrows in high-magnification images), mitochondria.





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Figure 6 | Nanoneedles mediate neovascularization. a, Intravitalbright-field (top) and confocal (bottom) microscopy images of thevasculature of untreated (left) and hVEGF-165-treated muscles with eitherdirect injection (centre) or nanoinjection (right). The fluorescence signaloriginates from systemically injected FITC–dextran. Scale bars, bright-field100 µm; confocal 50 µm. b,c, Quantification of the fraction of fluorescentsignal (dextran) (b) and the number of nodes in the vasculatureper mm2 (c) within each field of view acquired for untreated control,intramuscular injection (IM) and nanoinjection. ⇤p=0.05, ⇤⇤p<0.01,⇤⇤⇤p<0.001. Error bars represent the s.d. of the averages of 5 areas takenfrom 3 animals.

of the irregular neovasculature as observed with nanoinjection(Fig. 6a–c), but to the physiologic increased blood flow to the area.

The new blood vessels formed after VEGF nanoinjectionwere functional, and had blood flow rates comparable to pre-existing vessels of similar size (Supplementary Movies 1–3 andSupplementary Figs 15 and 16). The vasculature of treated musclesrevealed increased blood pooling, exhibiting both a greater numberof pools and a larger total pool size at 7 and 14 days (SupplementaryFig. 14). This increased blood pooling indicates the formationof immature, leaky capillaries, as expected in response to VEGFexpression41. Taken together, the data show that the nanoinjectionof VEGF modulated the local gene expression, favouring tissueneovascularization and increased local blood perfusion.

OutlookWe presented the synthesis and biomechanical characterizationof porous silicon nanoneedles. The needles were biocompatible,biodegradable and could e�ciently load and release nucleic acidsdirectly in the cytosol, negotiating local biological barriers (forexample, the cell membrane or the endolysosomal system). Thenanoinjection of di�erent nucleic acids could simultaneously up-or downregulate the expression of genes within a cell populationconfined in a region, thus enabling in vivo cell reprogrammingwithin selected areas of a tissue. We demonstrated as a proof of

concept that the nanoneedles mediated the in situ delivery of anangiogenic gene and triggered the patterned formation of new bloodvessels. This paves the way for the in vivo, local control of thephysiologic and structural features of a defined area of a tissueof interest.

The development of degradable nanoneedles highlights thetransition towards biodegradable micro- and nano structures ableto improve localized delivery while mitigating inflammation43.Microneedles are e�ective for three-dimensional delivery ofpayloads throughout tissues across hundreds of micrometres, andconsequently their ability to target only a superficial layer of cells isgreatly impaired44. Moreover, owing to the cell damage caused bytheir size, they induce local injury to the tissue, localized necrosis45,and generate responses at the tissue and systemic level (transientimmune activation)43. Conversely, nanoneedles are designed forextremely localized delivery to a few superficial layers of cells(two-dimensional patterning). This confined intracellular deliveryhas the potential to target specific exposed areas within a tissue,further reducing the invasiveness of the injection and limiting theimpact on the overall structure of the tissue. The confined natureof nanoinjection at present requires only a small surgical incision toaccess non-exposed sites (for example, muscles). Furthermore, thenanoneedles can be applied to tissues with di�erent structural andmechanical properties as a stepping stone towards the developmentof a non-immunogenic delivery platform for nucleic acids and otherfunctional payloads to tissues.

The nanoinjection of the angiogenic master regulator gene,VEGF165, enabled minimally invasive, localized in vivo geneticengineering of the muscle tissue. This form of delivery ushers incountless possibilities to design and study the microarchitectureof tissues and highlights a strategy for microscale modulation ofwidely diverse biological processes, ranging from immune responseto cell metabolism. In this study we focused on the induction ofneoangiogenesis in the muscle tissue, because early vascularizationis a key step in wound healing and scar-tissue remodelling.Moreover, the growth of a supporting blood-vessel network iscrucial for the successful grafting of an implant. Most sca�oldsemployed in tissue engineering rely on the host’s vasculature forthe supply of oxygen and nutrients. The nanoneedles could beused to pre-condition the site of an implant by locally patterninga supplemental vascular network. This could support the early stepsof tissue regeneration and, by enhancing the integration with thesurrounding native tissue, increase the long-term performance ofthe implant46.

MethodsFabrication of the nanoneedles. Low-stress silicon nitride (160 nm) wasdeposited by low-pressure chemical vapour deposition over 100mm, 0.01 �-cm,boron-doped p-type silicon wafers. A 600-nm-diameter disk array with desiredpitch was patterned on the substrate by photolithography, and transferred intothe silicon nitride layer by reactive ion etch in CF4 plasma.

Following photoresist stripping, the substrate was cleaned in 10% HF for 60 sfollowed by deposition of Ag from 1mM AgNO3 in 10% HF for 2min. Thesubstrate was rinsed in water and 2-propanol, and dried. Metal-assisted chemicaletch of the substrate in 10% HF, 122mM H2O2 for 8min 30 s formed poroussilicon pillars with 600 nm diameter and 9 µm height. The pillars were shapedinto nanoneedles with apical diameter <50 nm by reactive ion etch in SF6 plasma.The substrate was cut into 8 ⇥ 8mm chips, and the nanoneedles on the chipswere oxidized by O2 plasma.

Nanoinjection in vitro. 8 ⇥ 8mm nanoneedle chips were sterilized in 70%ethanol for 1 h and allowed to dry under ultraviolet irradiation. nN-B: The chipwas placed at the bottom of a 24-well plate, rinsed thrice with PBS, and 1 ⇥ 105HeLa cells were seeded over the needles. The well plate was returned to theincubator. nN-T: HeLa cells were seeded at 5 ⇥ 104 cells in a 24-well plate andincubated for 24–48 h. Three ml of fresh medium were added, and thenanoneedle chip was immersed in medium face down. The plate spun at 100 rcffor 1min in a swinging bucket centrifuge. For further culturing, the chip wasremoved from the well after 30min of incubation, and placed face up in a new24-well plate, with 0.5ml of fresh medium.






Delivery of nucleic acids in vitro. The nanoneedles were functionalized with3-aminopropyltriethoxysilane (APTES) by immersion for 2 h in 2% APTES inethanol. GAPDH cy3–siRNA (50 µl, 100 nM) in PBS was spotted on thenanoneedles chip and incubated for 15min, then rinsed three times with 100 µlof DI water. Nanoneedles were interfaced with cells either in the nN-T or nN-Bsettings. At 30min and 24 h following treatment, cells were fixed, mounted on acoverslip, and analysed by confocal microscopy. GAPDH silencing was assayed byin-cell western at 24 h following nN-B treatment. GAPDH protein expression wasquantified from the fluorescent intensity at 800 nm normalized to the total chipsurface area.

Alternatively, the chips were incubated with 20 µl of GAPDH cy3–siRNA(100 nM) and GFP plasmid (500 ng µl�1) in PBS. The solution was allowed to dryover the nanoneedles, and the needles were immediately employed in a nN-Bstrategy. For confocal microscopy the cells were fixed at 48 h followingtransfection. For flow-cytometry analysis the cells were trypsinized, collected in a1ml microcentrifuge tube, washed twice with PBS and then fixed.

Nanoinjection in vivo. Animal studies were performed in accordance with theguidelines of the Animal Welfare Act and the Guide for the Care and Use ofLaboratory Animals based on approved protocols by The University of TexasM.D. Anderson Cancer Center’s (MDACC) and Houston Methodist ResearchInstitute’s (HMRI) Institutional Animal Care and Use Committees. Immediatelybefore use, chips were cleaned, sterilized under ultraviolet light, and incubatedwith 20 µl of fluorescent dyes (1mgml�1) or pVEGF (5mgml�1) in PBS, whichwas allowed to dry.

Nanoneedles were pressed down using the thumb, ensuring minimalmovement for at least 1min, and then removed. For the muscle studies, a smallincision was made on the upper-back right of the mouse before application.

Plasmid VEGF delivery was performed with a 1–2 cm dorsal midlineincision to expose the superficial gluteal and lumbar muscles. VEGF-loadedpSi nanoneedles were implanted in direct contact with the lumbar and glutealmuscle on the right side, whereas the left side was left untreated. For the directinjection, 20 µl of VEGF (at 5mgml�1) were injected using a Hamilton Neuros25 µl syringe adjusted for a penetration depth of 200 µm to a similarlyexposed muscle.

Received 17 February 2014; accepted 11 February 2015;published online 30 March 2015

References1. Leader, B., Baca, Q. J. & Golan, D. E. Protein therapeutics: A summary and

pharmacological classification. Nature Rev. Drug Discov. 7, 21–39 (2008).2. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic therapeutic

peptides: Science and market. Drug Discov. Today 15, 40–56 (2010).3. Kole, R., Krainer, A. R. & Altman, S. RNA therapeutics: Beyond RNA

interference and antisense oligonucleotides. Nature Rev. Drug Discov. 11,125–140 (2012).

4. Khademhosseini, A. Microscale technologies for tissue engineering andbiology. Proc. Natl Acad. Sci. USA 103, 2480–2487 (2006).

5. Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down barriers:Advances in siRNA delivery. Nature Rev. Drug Discov. 8, 129–138 (2009).

6. Saltzman, W. M. & Luo, D. Synthetic DNA delivery systems. Nature Biotechnol.18, 33–37 (2000).

7. Thomas, C. E., Ehrhardt, A. & Kay, M. A. Progress and problems with the useof viral vectors for gene therapy. Nature Rev. Genet. 4, 346–358 (2003).

8. Mingozzi, F. & High, K. A. Therapeutic in vivo gene transfer for genetic diseaseusing AAV: Progress and challenges. Nature Rev. Genet. 12, 341–355 (2011).

9. Lefesvre, P., Attema, J. & van Bekkum, D. A comparison of e�cacy and toxicitybetween electroporation and adenoviral gene transfer. BMCMol. Biol. 3,12 (2002).

10. Aihara, H. & Miyazaki, J-I. Gene transfer into muscle by electroporationin vivo. Nature Biotechnol. 16, 867–870 (1998).

11. Nishikawa, M. & Huang, L. Nonviral vectors in the new millennium: Deliverybarriers in gene transfer. Hum. Gene Ther. 12, 861–870 (2001).

12. Shalek, A. K. et al. Vertical silicon nanowires as a universal platform fordelivering biomolecules into living cells. Proc. Natl Acad. Sci. USA 107,1870–1875 (2010).

13. Robinson, J. T. et al. Vertical nanowire electrode arrays as a scalable platformfor intracellular interfacing to neuronal circuits. Nature Nanotech. 7,180–184 (2012).

14. Yan, R. et al. Nanowire-based single-cell endoscopy. Nature Nanotech. 7,191–196 (2011).

15. Han, S. W. et al.High-e�ciency DNA injection into a single humanmesenchymal stem cell using a nanoneedle and atomic force microscopy.Nanomedicine: Nanotechnol. Biol. Med. 4, 215–225 (2008).

16. Shalek, A. K. et al. Nanowire-mediated delivery enables functionalinterrogation of primary immune cells: Application to the analysis of chroniclymphocytic leukemia. Nano Lett. 12, 6498–6504 (2012).

17. Xu, A. M. et al. Quantification of nanowire penetration into living cells. NatureCommun. 5, 3613 (2014).

18. Sharei, A. et al. A vector-free microfluidic platform for intracellular delivery.Proc. Natl Acad. Sci. USA 110, 2082–2087 (2013).

19. Hanson, L., Lin, Z. C., Xie, C., Cui, Y. & Cui, B. Characterization of thecell–nanopillar interface by transmission electron microscopy. Nano Lett. 12,5815–5820 (2012).

20. Xie, X. et al.Mechanical model of vertical nanowire cell penetration. Nano Lett.13, 6002–6008 (2013).

21. Xie, C., Lin, Z., Hanson, L., Cui, Y. & Cui, B. Intracellular recording of actionpotentials by nanopillar electroporation. Nature Nanotech. 7, 185–190 (2012).

22. Wang, Y. et al. Poking cells for e�cient vector-free intracellular delivery. NatureCommun. 5, 4466 (2014).

23. Anderson, S. H. C., Elliott, H., Wallis, D. J., Canham, L. T. & Powell, J. J.Dissolution of di�erent forms of partially porous silicon wafers undersimulated physiological conditions. Phys. Status Solidi A 197, 331–335 (2003).

24. Canham, L. T. Bioactive silicon structure fabrication through nanoetchingtechniques. Adv. Mater. 7, 1033–1037 (1995).

25. Chiappini, C. et al. Tailored porous silicon microparticles: Fabrication andproperties. ChemPhysChem 11, 1029–1035 (2010).

26. Tasciotti, E. et al.Mesoporous silicon particles as a multistage delivery systemfor imaging and therapeutic applications. Nature Nanotech. 3, 151–157 (2008).

27. Tanaka, T. et al. Sustained small interfering RNA delivery by mesoporoussilicon particles. Cancer Res. 70, 3687–3696 (2010).

28. Park, J-H. et al. Biodegradable luminescent porous silicon nanoparticles forin vivo applications. Nature Mater. 8, 331–336 (2009).

29. Goh, A. S-W. et al. A novel approach to brachytherapy in hepatocellularcarcinoma using a phosphorous32 (32P) brachytherapy delivery device—afirst-in-man study. Int. J. Radiat. Oncol. Biol. Phys. 67, 786–792 (2007).

30. Anglin, E., Cheng, L., Freeman, W. & Sailor, M. Porous silicon in drug deliverydevices and materials. Adv. Drug Deliv. Rev. 60, 1266–1277 (2008).

31. Chiappini, C., Liu, X., Fakhoury, J. R. & Ferrari, M. Biodegradable poroussilicon barcode nanowires with defined geometry. Adv. Funct. Mater. 20,2231–2239 (2010).

32. Obataya, I., Nakamura, C., Han, S., Nakamura, N. & Miyake, J. Mechanicalsensing of the penetration of various nanoneedles into a living cell using atomicforce microscopy. Biosens. Bioelectron. 20, 1652–1655 (2005).

33. Na, Y-R. et al. Probing enzymatic activity inside living cells using ananowire–cell ‘sandwich’ assay. Nano Lett. 13, 153–158 (2013).

34. Muralidharan-Chari, V., Clancy, J. W., Sedgwick, A. & D’Souza-Schorey, C.Microvesicles: Mediators of extracellular communication during cancerprogression. J. Cell Sci. 123, 1603–1611 (2010).

35. Ratajczak, J. et al. Embryonic stem cell-derived microvesicles reprogramhematopoietic progenitors: Evidence for horizontal transfer of mRNA andprotein delivery. Leukemia 20, 847–856 (2006).

36. Yuan, A. et al. Transfer of microRNAs by embryonic stem cell microvesicles.PLoS ONE 4, e4722 (2009).

37. Shen, Z. et al. Porous silicon as a versatile platform for laserdesorption/ionization mass spectrometry. Anal. Chem. 73, 612–619 (2000).

38. Lee, J. W., Park, J-H. & Prausnitz, M. R. Dissolving microneedles fortransdermal drug delivery. Biomaterials 29, 2113–2124 (2008).

39. Gross, S. et al. Bioluminescence imaging of myeloperoxidase activity in vivo.Nature Med. 15, 455–461 (2009).

40. Luxembourg, A., Evans, C. F. & Hannaman, D. Electroporation-based DNAimmunisation: Translation to the clinic. Expert Opin. Biol. Ther. 7,1647–1664 (2007).

41. Yancopoulos, G. D. et al. Vascular-specific growth factors and blood vesselformation. Nature 407, 242–248 (2000).

42. Tammela, T., Enholm, B., Alitalo, K. & Paavonen, K. The biology of vascularendothelial growth factors. Cardiovasc. Res. 65, 550–563 (2005).

43. DeMuth, P. C. et al. Polymer multilayer tattooing for enhancedDNA vaccination. Nature Mater. 12, 367–376 (2013).

44. Kim, Y-C., Park, J-H. & Prausnitz, M. R. Microneedles for drug and vaccinedelivery. Adv. Drug Deliv. Rev. 64, 1547–1568 (2012).

45. Gershonowitz, A. & Gat, A. VoluDermMicroneedle Technology for SkinTreatments—In VivoHistological Evidence. J. Cosmetic Laser Surg. 17,9–14 (2015).

46. Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H. & Tomic-Canic, M.Growth factors and cytokines in wound healing.Wound Repair Regen. 16,585–601 (2008).

AcknowledgementsThe authors would like to thank M. Giacca at the International Centre for GeneticEngineering and Biotechnology (ICGEB in Trieste, Italy) for kindly donating thepDNA-expressing VEGF-165, and I. K. Yazdi and R. Palomba for the amplification andpurification of the plasmids used in the study. We are grateful to S. Amra forhistology-slide preparation, D. Tinkey for surgical procedures, S. Zacchigna forsuggestions on PCR protocols, P. Campagnolo for advice on SMA and isolectin staining,and S. T. Gammon for advice on bioluminescence imaging with luminol. This work wasfinancially supported by: the US Department of Defense (W81XWH-12-10414) and theNIH (1R21CA173579-01A1 and 5U54CA143837); C.C. was supported by a Newton





NATUREMATERIALS DOI: 10.1038/NMAT4249 ARTICLESInternational Fellowship and a Marie Curie International Incoming Fellowship; J.O.M.was supported by a NIH pre-doctoral fellowship 5F31CA154119-02. M.M.S. holds anERC grant ‘Naturale-CG’ and is supported by a Wellcome Trust Senior InvestigatorAward. E.T. holds grants from the Hearst Foundation and the Cullen Trust Foundation.C.C. and M.M.S. thank the Rosetrees Trust and the Stoneygate Trust for funding.

Author contributionsC.C., E.T. and M.M.S. designed the research. C.C. and E.T. conceived the nanoneedles;C.C. developed the nanoneedles with contributions from X.L.; C.C. evaluated loading,release, delivery and e�cacy with contributions from E.D.R.; C.C. imagedbiodegradation and cell interaction with contributions from E.D.R.; C.C. wrote the initialmanuscript with contributions from E.D.R. and J.O.M. C.C. performed all electronmicroscopy analysis. E.D.R. and J.O.M. assessed cytocompatibility; E.D.R. designed andperformed animal surgeries, intravital imaging and quantification of vascularization,vasculature pattern, and extravasation and flow rate over time on VEGF treatment, with

contributions from J.O.M. J.O.M. evaluated degradation by ICP-AES, fluorescent andbioluminescent imaging and analysis, real-time PCR, and histological evaluation andstaining of tissues with contributions from E.D.R. J.S. performed compressive mechanicaltesting. E.T. and M.M.S. contributed equally to the work, with M.M.S. supervising thein vitro studies and E.T. the in vivo work. All authors discussed and commented onthe manuscript.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints.Correspondence and requests for materials should be addressed to M.M.S. or E.T.

Competing financial interestsThe authors declare no competing financial interests.





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