Journal of Nanomedicine & Biotherapeutic Discovery
Open Access

Biodegradable Silicon Nanoneedles for Intracellular Delivery of CRISPR Components in Genetic Skin Disorders

Xuan He^{* *}Correspondence: Xuan He, Department of Biomedical Engineering, Peking University, Beijing, China, Email:

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Description

Inherited skin disorders, including epidermolysis bullosa and pachyonychia congenita, represent significant therapeutic challenges due to the complexity of delivering gene editing components to the epidermis while navigating its natural barrier properties. We have developed biodegradable silicon nanoneedles capable of efficiently delivering Clustered Regularly Interspaced Short Palindromic Repeats associated Protein 9 (CRISPR-Cas9) Ribonucleoproteins (RNPs) directly to epidermal keratinocytes, enabling precise genetic correction with minimal disruption to skin architecture. These high-aspect-ratio nanoneedles, measuring approximately 10μm in length with 150nm diameter tips, facilitate direct cytosolic delivery of macromolecular cargos while minimizing cellular damage and inflammatory responses.

The nanoneedles were fabricated through reactive ion etching of monocrystalline silicon, creating precisely controlled geometries optimized for keratinocyte penetration. Surface modification with dopamine-conjugated polyethylene glycol created a versatile coating for biomolecule attachment while imparting favorable tissue interaction properties. The silicon backbone was engineered with precisely controlled porosity through electrochemical etching, creating a structure that undergoes gradual dissolution in physiological environments with complete degradation occurring within approximately 2 weeks. CRISPR components, including Cas9 protein and guide RNAs targeting specific mutations in the KRT14 gene implicated in epidermolysis bullosa simplex, were attached to the nanoneedle surface through biodegradable linkers that facilitate release following cellular entry.

In vitro characterization using patient-derived keratinocytes demonstrated efficient delivery, with approximately 87% of cells showing successful penetration without significant impact on viability as assessed by live/dead staining. Confocal microscopy confirmed direct cytosolic delivery of fluorescently labeled RNPs, bypassing endosomal entrapment that often limits conventional delivery approaches. Genome editing efficiency assessed through next-generation sequencing revealed approximately 64% correction of the disease-causing mutation, with indel formation at predicted off-target sites remaining below 0.3%. Functional restoration was confirmed through immunofluorescence staining showing normalized keratin 14 filament organization and mechanical stress testing demonstrating improved resilience to physical deformation compared to uncorrected cells.

Ex vivo studies utilizing three-dimensional human skin equivalents generated from patient keratinocytes demonstrated successful penetration of nanoneedles throughout the epidermis following application with a custom microneedle applicator device. Spatial mapping of editing events confirmed correction throughout all epidermal layers, with particularly efficient editing in basal keratinocytes — the progenitor population critical for sustained therapeutic effect. Following growth and differentiation of treated skin equivalents, immunohistochemical analysis revealed normalized tissue architecture with proper basement membrane formation and restored expression of structural proteins critical for skin integrity.

In vivo evaluation utilized a humanized mouse model where patient-derived skin was grafted onto immunodeficient mice, creating an accessible system for testing therapeutic approaches. Application of nanoneedle arrays to established grafts resulted in successful penetration throughout the epidermis, with minimal inflammatory response as assessed by cytokine profiling and histological examination. Molecular analysis of treated grafts demonstrated approximately 48% correction of the disease-causing mutation at 4 weeks post-treatment, with edited cells showing proper integration within the tissue and contribution to ongoing epidermal renewal. Clinical assessment demonstrated significant improvements in mechanical integrity, with treated grafts showing resistance to blister formation following standardized friction testing.

Conclusion

Safety evaluation through comprehensive histopathological analysis confirmed normal wound healing responses following nanoneedle application, with complete re-establishment of barrier function within 24 hours as assessed by transepidermal water loss measurements. Silicon biodegradation products were efficiently cleared without evidence of accumulation or toxicity. Importantly, no evidence of immune responses against Cas9 protein was detected, likely due to the transient nature of the delivery system. These biodegradable silicon nanoneedles represent a promising approach for targeted gene editing in the epidermis, potentially enabling treatment of various genetic skin disorders through precise correction of causative mutations in keratinocyte progenitors capable of sustained therapeutic effect.