Flexible elastomer patch with vertical silicon nanoneedles for intracellular and intratissue nanoinjection of biomolecules

Abstract

Vertically ordered arrays of silicon nanoneedles (Si NNs), due to their nanoscale dimension and low cytotoxicity, could enable minimally invasive nanoinjection of biomolecules into living biological systems such as cells and tissues. Although production of these Si NNs on a bulk Si wafer has been achieved through standard nanofabrication technology, there exists a large mismatch at the interface between the rigid, flat, and opaque Si wafer and soft, curvilinear, and optically transparent biological systems. Here, we report a unique methodology that is capable of constructing vertically ordered Si NNs on a thin layer of elastomer patch to flexibly and transparently interface with biological systems. The resulting outcome provides important capabilities to form a mechanically elastic interface between Si NNs and biological systems, and simultaneously enables direct imaging of their real-time interactions under the transparent condition. We demonstrate its utility in intracellular, intradermal, and intramuscular nanoinjection of biomolecules into various kinds of biological cells and tissues at their length scales.

Fig. 1. Images and illustrations for the integration of vertically ordered Si NNs onto an elastomer patch.

(A) A series of scanning electron microscopy (SEM) images of vertically ordered Si pillars with selected passivation layer (left), with localized undercut (middle), and after the size is reduced down to the nanoscale (right). Scale bar, 1 μm. (B) Schematic illustrations of the key steps to physically liberate Si NNs from their native Si wafer via the swelling of PDMS. (C) Optical image of a representative Si NN-patch. Scale bar, 1.5 cm. (D) Magnified SEM image of the partly embedded Si NNs into PDMS. The inset highlights the needle-like sharp tips. Scale bars, 20 μm and 600 nm (inset). (E) Confocal laser scanning microscopy (CLSM) image of Si NNs. Scale bar, 30 μm.

Fig. 2. Mechanism study and mechanical analysis for controlled cracking of Si NNs.

(A) FEA results of displacement of PDMS under swelling at 100, 170, and 230%. (B) Corresponding FEA results of maximum principal strain distributions along a single Si NN during each swelling condition. (C) Computational data showing the effect of peak strain (ε_peak) on S (left), D/d ratio (middle), and H/h ratio (right). Black dashed line denotes a theoretical fracture limit of the Si NN.

Fig. 3. Basic characterizations of Si NN-patch.

(A) Results of MTT assay in the cytotoxicity tests of HDF cells interfaced with the Si NN-patch (green) and control Si NNs on a Si wafer (red) and a bare Si wafer (yellow). Error bar represents the SD of three replicates. (B) Results of LDH assay in the invasiveness tests of HDF cells for 2 days. Triton X-100 is used as a positive control (blue). Error bar represents the SD of three replicates. (C) SEM images of MCF7 cells on a representative Si NN-patch at 24 hours after nanoinjection. Scale bar, 10 μm. Arrows highlight the deformed Si NNs by cell deformations. (D) Time-lapsed live differential interference contrast (DIC) images of HDF cells that interacted with Si NNs at the bottom. Scale bar, 10 μm.

Fig. 4. Formation of nanoscale surface pores and intracellular nanoinjection of siRNA.

(A) SEM images of nanopores formed on the surface of Si NNs at different treatment times of MACE. Scale bar, 250 nm. (B) Confocal microscopy images of Si NNs with (left) and without (right) nanopores on the surface by using a green fluorescence dye. Scale bar, 15 μm. (C) Confocal microscopy image of nanoporous Si NNs loaded with Cy3-siRNAs (red). Scale bar, 15 μm. (D) Confocal microscopy image of GFP-MCF7 cells at 24 hours after nanoinjection of Cy3-siRNAs. Scale bar, 15 μm. (E) Results of flow cytometry (FACS) analysis for SKOV3 cells at 48 hours after nanoinjection of Cy3-siRNAs by using the nanoporous Si NN-patch (green) and control Si NNs on a bulk Si wafer with (red) and without (blue) nanopores on the surface and a bare Si wafer (yellow). (F) Corresponding results of GAPDH analysis for the SKOV3 cells. Error bar represents the SD of three replicates.

Fig. 5. Intratissue nanoinjection of Si NN-patch.

(A) Optical images of the mice interfaced with the Si NN-patch for intradermal (left) and intramuscular (middle) nanoinjection. Scale bar, 1 cm. Conventional Si NNs built on a bulk Si wafer are used for control comparison (right). (B) Corresponding IVIS images of the mice for up to 48 hours after nanoinjection. Scale bar, 2 cm.

Fig. 6. In vivo tissue compatibility of Si NN-patch.

(A) Real-time bioluminescence images on the skin, muscle, and ear of the mice at 5 hours following the implementation of the Si NN-patch (left column) and control Si NNs (middle column) and with control PMA treatment (right column). Scale bar, 10 mm. (B) Corresponding H&E histological cross-sectional views of the treated tissue sections. Scale bar, 400 μm.