Potential Therapeutic Applications of Spherical Nucleic Acids (SNAs) in Cancer: Key Observations and Insights

Abstract

Spherical nucleic acids (SNAs) are a unique class of nucleic acid nanotherapeutics characterized by densely packed, radially arranged oligonucleotides surrounding a nanoparticle or molecular core, or core-free nanoscale structures. Compared with conventional linear nucleic acids, SNAs exhibit excellent cellular uptake, resistance to nuclease degradation, and efficient target recognition without the help of transfection agents. This review summarizes SNAs’ structural diversity, design principles, and the multiscale delivery barriers they encounter. We mainly focus on liposomal SNAs (LSNAs) as programmable platforms for cancer immunotherapy. We emphasize how lipid chemistry, membrane architecture, and anchor stability control lymphoid distribution, immune-cell uptake, antigen presentation, and strong antitumor immunity, rather than seeing lipids as inert carriers. Furthermore, we also discuss current developments in gene silencing, CRISPR-based genome editing, and stimulus-responsive multimodal therapies, highlighting the significant therapeutic advancements of SNAs like clinical trial NU-0129 and LSNAs like cavrotolimod. Finally, we describe present translational obstacles and potential prospects by using SNAs as next-generation precision immunotherapeutics.

Introduction

Despite remarkable advances in surgery, radiotherapy, chemotherapy, immunotherapy, and precision medicine,^1,2 cancer remains the second leading cause of death worldwide.³ Among these treatments, nucleic acid therapeutics, including antisense oligonucleotides (ASOs),⁴ small interfering RNAs (siRNAs),⁵ microRNAs (miRNAs),⁶ messenger RNAs (mRNAs),⁷ plasmid DNAs (pDNAs),⁸ aptamers,⁹ and CRISPR/Cas gene-editing systems,¹⁰ have enabled sequence-specific modulation of tumor-associated genes and signaling pathways. However, linear nucleic acids are rapidly degraded by circulating nucleases, exhibit poor membrane permeability because of their hydrophilic nature and negative charge, and may undergo rapid renal clearance or induce undesirable immune responses.^11,12 Uptake and clearance in organs like the liver and spleen affect the biodistribution of oligonucleotides after systemic injection, which may restrict their delivery to tumor tissues and cells.¹³ These biological barriers can therefore limit the therapeutic of oligonucleotide-based therapies.

To overcome these limitations, diverse delivery platforms, such as viral vectors, lipid nanoparticles (LNPs), polymeric nanoparticles, extracellular vesicles, and inorganic nanocarriers, have been developed.^14–16 Among these systems, LNPs have emerged as a leading platform for non-viral nucleic acid delivery following the clinical success of mRNA vaccines and the approval of the first LNP-based siRNA therapeutic.^17–19 Nevertheless, current LNP technologies still exhibit several intrinsic limitations, including predominant liver accumulation, limited extrahepatic delivery, insufficient cellular specificity within heterogeneous tumor tissues, and variable endosomal escape efficiency.²⁰ These limitations have shifted research from simply improving delivery efficiency toward engineering the biological fate of nanocarriers, including organ tropism, cellular specificity, intracellular trafficking, and ultimately therapeutic precision.

In this context, spherical nucleic acids (SNAs) have emerged as a uniquely potent platform, characterized by a dense, three-dimensional (3D) arrangement of oligonucleotides around a nanoparticle core or within a self-assembled nanoscale architecture.²¹ Unlike conventional linear nucleic acids, the distinctive spherical architecture of SNAs helps to protect oligonucleotides from nuclease degradation and promotes cellular uptake without conventional transfection reagents. The reason for cellular internalization is partly due to class A scavenger receptors.²² But efficient cellular internalization does not ensure therapeutic success. To improve anticancer efficacy, systemically administered SNAs must overcome several biological barriers. These include clearance from circulation, limited access to target tissues, and inefficient uptake by the intended cell populations. Additional challenges depend on the therapeutic application, such as penetration into solid tumors or endosomal escape for payloads that act in the cytosol or nucleus. Therefore, engineering SNAs to achieve multiscale targeting-spanning organ, tissue, cellular, and subcellular levels-has become a central objective in the development of next-generation nucleic acid nanomedicines.^23,24

While our previous publication provided a broad overview of diverse smart nanocarriers, such as lipid nanoparticles, extracellular vesicles, and polymers, for delivering general therapeutic nucleic acids in cancer immunotherapy,²⁵ the present review narrows its scope specifically to SNAs. Furthermore, recent reviews summarized SNA structures and their general applications.^26,27 Here, we discuss how SNA design influences biological behavior and therapeutic activity, with particular attention to liposomal SNAs (LSNAs) for cancer immunotherapy. We also discuss delivery barriers at the organ, tissue, cellular, and subcellular levels, along with the challenges facing clinical translation (Figure 1).

Figure 1.

Overview of SNA structural engineering, biological fate, therapeutic applications, and clinical translation. SNA structure influences circulation, tissue distribution, cellular uptake, and intracellular trafficking. Applications include gene silencing, genome editing, cancer immunotherapy, and stimulus-responsive combination therapy. NU-0129 and cavrotolimod (AST-008) are examples of clinical investigations. SNA structures were drawn with assistance from ChatGPT (GPT-4o, OpenAI) and subsequently edited and assembled by the authors.

Evolution and Structural Engineering of SNAs

As described above, SNAs are defined primarily by their ordered three-dimensional architecture, which generally consists of a multifunctional core densely functionalized with a radially oriented shell of single-stranded oligonucleotides. In many core-anchored SNAs, the surface oligonucleotides are approximately 25–40 nucleotides long and contain three functional domains: an attachment group, a spacer region, and a recognition sequence.²⁸ The attachment group, commonly modified with alkyl thiol, cyclic disulfide, or other reactive chemical groups, enables the conjugation of oligonucleotides to the nanoparticle surface. Between the attachment group and the functional sequence, a spacer region, typically composed of oligo (ethylene glycol) (OEG), ie, a short oligomeric analogue of polyethylene glycol (PEG), or a short poly(T) or poly(A) sequence (5 −10 nucleotides), is frequently introduced to improve chain flexibility, reduce steric hindrance, and enhance structural stability.^29,30 The terminal recognition sequence is designed to hybridize with complementary nucleic acid targets or serve as the therapeutic moiety^31,32 (Figure 2A).

Figure 2.

Structural organization, core-based classification, and representative architectures of SNAs. (A) Basic SNA, consisting of a multifunctional core surrounded by a dense, radially oriented oligonucleotide shell with an attachment group, a spacer region, and a recognition sequence. (B) Classification of SNAs according to the nature or absence of the core, including inorganic-core SNAs, organic-core SNAs, and core-free SNAs. Selected SNA structures were drawn with assistance from ChatGPT (GPT-4o, OpenAI) and subsequently edited and assembled by the authors.

The exceptional structural programmability of SNAs has enabled continuous evolution of their core materials and assembly strategies. Based on the core composition, SNAs are broadly classified into three major classes. More recently, the concept of molecular spherical nucleic acids (m-SNAs) has further expanded the structural precision of SNAs by allowing exact control over both the number and spatial arrangement of oligonucleotides on molecular-scale cores such as polyhedral oligomeric silsesquioxane (POSS) and fullerene (C60).^33–35 Unlike conventional nanoparticle-based SNAs that display a statistical average of surface strands, m-SNAs provide absolute molecular definition with a discrete number of conjugated oligonucleotides, thus representing a structurally precise subclass.

Structural Organization and Compositional Diversity

Since their first report in 1996, SNAs have provided a distinctive approach to addressing the poor stability and inefficient cellular uptake associated with conventional linear nucleic acids.^21,36 Based on the nature or absence of the core, they are broadly divided into inorganic-core SNAs, organic-core SNAs, and core-free SNAs (Figure 1B). These classifications represent different design strategies that continue to develop at the same time. AuNPs remain the most widely used inorganic cores because of their high stability and ease of surface modification, especially for biosensing and the in vitro detection of biomarkers in serum.^37,38 Besides AuNPs, a variety of inorganic materials, including silver (Ag),³⁹ platinum (Pt),³⁹ iron oxide (Fe₃O₄),⁴⁰ and silica cores,⁴¹ have subsequently been incorporated into SNA platforms to endow additional optical, magnetic, catalytic, or therapeutic functionalities. Despite their excellent structural stability and facile surface functionalization, some persistent inorganic-core SNAs may undergo prolonged tissue retention, thereby raising concerns regarding biosafety and limiting their clinical translation.

Organic- and soft-matter-core SNAs extend the range of available SNA materials through the use of liposomal,⁴¹ polymeric,⁴² and protein-based cores.⁴³ Many soft-matter systems offer improved biodegradability, structural flexibility, and cargo-loading capacity compared with persistent metallic cores. In 2014, Mirkin et al reported the first LSNAs, in which oligonucleotides were densely assembled on the surface of liposomes, enabling efficient transfection-agent-free cellular uptake and potent antisense gene silencing without auxiliary transfection reagents.⁴¹ Subsequently, protein SNAs (ProSNAs) were developed by covalently conjugating oligonucleotides onto β-galactosidase through click chemistry, resulting in approximately 280-fold higher cellular uptake while maintaining enzymatic activity at picomolar concentrations.⁴⁴ Around the same period, polymeric SNAs (PSNAs) based on DNA brush block copolymers (DBBCs) were introduced, providing biodegradable scaffolds with improved cargo-loading capacity and structural flexibility.⁴⁵ In specific designs, the core can additionally serve as a therapeutic cargo. Nevertheless, soft-matter SNAs may still face challenges related to preparation, structural stability, and manufacturing reproducibility remain important challenges.

Core-free SNAs have also been developed to reduce the limitations associated with permanent or inactive carriers. Hollow SNAs are typically fabricated by first assembling oligonucleotides onto sacrificial AuNP templates followed by selective core removal, generating stable hollow nanostructures that retain a highly oriented nucleic acid shell while avoiding permanent inorganic residues.³³ In contrast, carrier-free SNAs are constructed entirely through the self-assembly of nucleic acid therapeutics or amphiphilic nucleic acid conjugates without requiring preformed nanoparticle scaffolds. Zhu et al⁴⁶ constructed a carrier-free SNA through the self-assembly of paclitaxel (PTX)-oligonucleotide conjugates, in which two PTX molecules were linked to a fluorodeoxyuridine(FdU)-containing antisense oligonucleotide through a dithiomaleimide linker. This design maximizes the proportion of therapeutically active components and molecular programmability while eliminating carrier-associated toxicity. Ultimately, rather than representing a linear transition, these architectures continue to evolve in parallel to provide complementary advantages in stability, biodegradability, loading capacity, and structural precision (Figure 2B).

Beyond core classification, the structural scaffold and tunable biological interface of SNAs provide a basis for tailoring their interactions with immune cells and cancer cells.^25,32 In SNA vaccine designs, the scaffold enables the spatial organization of antigens and nucleic acid adjuvants,⁴⁷ while the biological interface influences cellular uptake and engagement of immune receptors.^22,24,48 Together, these features help coordinate antigen delivery and immune stimulation within antigen-presenting cells, supporting subsequent T-cell responses against cancer cells.^49,50 Surface targeting ligands can alternatively promote direct uptake by cancer cells.⁵¹ Although these SNA-specific scaffold and interface properties also influence chemotherapeutic delivery and radiosensitization,^52,53 their additional roles in coordinating immune activation and antigen presentation warrant particular attention in cancer immunotherapy. These mechanisms and their therapeutic implications are discussed in the cancer immunotherapy section.

Synthetic and Assembly Strategies

The nucleic acid shell can be connected to the SNA core via surface covalent conjugation, noncovalent anchoring, coordination, or molecular self-assembly,⁵⁴ which collectively govern stability, oligonucleotide density, and biological activity (Table 1). The selected method depends on the surface chemistry of the core and affects the stability, oligonucleotide density, orientation, and biological activity of SNAs.⁵⁵ For lipid-based SNAs, the anchoring strategy is especially consequential because it determines oligonucleotide retention, surface density, orientation, accessibility to pattern-recognition receptors, and release kinetics.⁵⁶ However, these biodistribution patterns are dependent on different cholesterol, diacylglycerol, phospholipid, and cleavable lipid anchors, and do not necessarily apply to all SNA architectures.⁵⁷

Table 1.

Connection Mechanisms Between SNA Cores and Oligonucleotide Shells

Assembly Strategy	Connection Mechanism	Typical Linkage or Interaction	Representative SNA System	Reference
Metal-thiol anchoring and coordination	Au-S conjugation	Thiolated RelA aptamers attached to 13 nm AuNP core via Au-S bonds	AuNP-aptamer SNA (E2-SNA)	[58]
	Multidentate cyclic disulfide-metal anchoring	Triple cyclic disulfide-modified oligonucleotides multivalently anchored onto AgNP surfaces via Ag-S interactions	DNA-Ag NPs	[59]
	Metal-phosphate coordination	Fe3⁺ in metal-phenolic network core coordinated with phosphate groups of oligonucleotide backbone	Fe-Shik@Fe-AS1411 and Fe-Gal@Fe-CpG	[60]
Covalent conjugation and cleavable linkers	Cleavable linker-mediated covalent conjugation	siRNA and pASO coupled via ROS-sensitive linker, followed by carrier-free self-assembly	Photolabile SNA	[61]
	Peptide-mediated covalent conjugation	Thiolated CpG coupled to DOPE via peptide linker (disulfide and amide bonds)	DOX/CpG-loaded liposomal SNA	[62]
	Amide coupling (EDC/NHS chemistry)	Carboxyl-functionalized polymeric nanoparticles coupled to 3′-amine-terminated oligonucleotides	Dox-PSNAs	[63]
	Thiol-maleimide conjugation	5′-thiol-modified DNA coupled to maleimide-terminated DPPE-PEG lipids via thiol-Michael addition	CRISPR LNP-SNA	[64]
Hydrophobic insertion and DNA hybridization	Hydrophobic insertion	5′-cholesterol-modified primer strands inserted into exosomal lipid membrane	Cholesterol-anchored exosome-DNA SNA	[38]
	DNA hybridization	Watson-Crick base pairing between palindromic tails of homopolymerized DNA core and connection arms of tripod DNA shell	ApRz-CS/Dox	[65]
	Hydrophobic insertion and DNA hybridization	3′-cholesterol-terminated CpG strands inserted into DOPC liposomes, followed by hybridization with antigen-DNA conjugates	Dual-antigen SNA vaccine (DA-SNA 1 and 2)	[47]
	Covalent DNA-hydrophobic block conjugation	19-mer DNA strand coupled to 12 dodecane units (phosphoramidite chemistry), followed by amphiphilic self-assembly	BKM120-loaded HE12-SNA	[66]
Click chemistry	Copper-catalyzed click conjugation (CuAAC)	Terminal alkyne-modified oligonucleotides coupled to azide-functionalized SPION cores	DNA–SPION conjugates	[40]
	Copper-free click conjugation (SPAAC)	DBCO-modified oligonucleotides coupled to azide-functionalized PLA/PEG	Bcl-2 ASO/HIF-1α siRNA-loaded PLA-PEG SNA	[67]
	SPAAC and DNA hybridization	DBCO-modified Bcl-2 ASOs coupled to azide-functionalized PLA, followed by hybridization with complementary DNA hairpins	polymeric micelle SNA (Ep-SNA@DOX)	[42]

Abbreviations: AgNP, silver nanoparticle; ASO, antisense oligonucleotide; AuNP, gold nanoparticle; Bcl-2, B-cell lymphoma 2; BKM120, buparlisib; CpG, cytosine-phosphate-guanine; CRISPR, clustered regularly interspaced short palindromic repeats; CuAAC, copper-catalyzed azide-alkyne cycloaddition; DBCO, dibenzocyclooctyne; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOX, doxorubicin; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HIF-1α, hypoxia-inducible factor 1-alpha; LNP, lipid nanoparticle; MMP, matrix metalloproteinase; NHS, N-hydroxysuccinimide; pASO, peptide nucleic acid-based antisense oligonucleotide; PEG, poly(ethylene glycol); PLA, polylactic acid; PSNA, photolabile/polymeric spherical nucleic acid; ROS, reactive oxygen species; siRNA, small interfering RNA; SNA, spherical nucleic acid; SPAAC, strain-promoted azide-alkyne cycloaddition; SPION, superparamagnetic iron oxide nanoparticle.

Metal-Thiol Anchoring and Coordination

Metal-thiol interactions robustly anchor thiolated oligonucleotides onto nanoparticle surfaces to form compact polynucleotide layers. In the classical AuNP-based system, thiolated RelA aptamers were attached to a 13 nm AuNP core to construct an apurinic/apyrimidinic endonuclease 1 (APE1)-responsive AuNP-aptamer SNA (E2-SNA).⁵⁸ This strategy has been extended to other noble-metal cores, such as DNA-silver nanoparticle conjugates where oligonucleotides containing three cyclic disulfide groups provide greater interfacial stability through multidentate interactions.⁵⁹ Beyond metal-thiol interactions, metal ions such as Fe³⁺ within a metal-phenolic network core can directly coordinate with the oligonucleotide phosphate backbone, enabling shell formation without terminal reactive groups.⁶⁰

Covalent Conjugation and Cleavable Linkers

For cores lacking suitable metal-binding surfaces, oligonucleotides are incorporated via functional linkers or covalent reactions. In photolabile SNAs, siRNA and a peptide nucleic acid-based antisense oligonucleotide are covalently connected through a singlet-oxygen-cleavable linker for carrier-free self-assembly.⁶¹ In Doxorubicin (DOX)/Cytosine-phosphate-guanine (CpG)-loaded LSNAs, thiolated CpG is connected to a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipid anchor through a peptide containing disulfide and amide linkages, thereby enabling Matrix metalloproteinase 9 (MMP9)-responsive cleavage.⁶² Direct core-oligonucleotide conjugation is also achieved through conventional amide-bond formation, such as coupling carboxyl groups on polymeric nanoparticles with amine-terminated oligonucleotides using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry.⁶³ Thiol-maleimide chemistry provides another efficient route, utilized in CRISPR LNP-SNAs to couple thiol-modified DNA with maleimide-terminated 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DPPE-PEG) lipids, resulting in a compact multivalent DNA layer.⁶⁴

Hydrophobic Insertion and DNA Hybridization

Lipid-containing cores support hydrophobic anchoring, allowing cholesterol-modified primer strands to be inserted directly into exosomal or liposomal membranes.³⁸ DNA hybridization provides programmable organization for preassembled components. For example, Watson-Crick base pairing between the palindromic tails of the homopolymerized DNA core and a tripod DNA shell generated an AS1411 aptamer- and pistol ribozyme-anchored DNA core-shell SNA.⁶⁵ These methods can be employed sequentially, such as inserting cholesterol-terminated CpG strands into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes followed by hybridization with complementary antigen-DNA conjugates to construct dual-antigen vaccines.⁴⁷ Alternatively, the core can be generated through the self-assembly of covalently synthesized DNA hydrophobic block conjugates. Additionally, covalently synthesized amphiphilic conjugates, like a 19-mer DNA strand coupled with dodecane units, can self-assemble directly into buparlisib (BKM120) -loaded core-free SNAs.⁶⁶

Click Chemistry

Click chemistry expands the range of functionalizable core materials.⁶⁸ Copper-catalyzed azide-alkyne cycloaddition (CuAAC) efficiently couples alkyne-modified oligonucleotides with azide-functionalized superparamagnetic iron oxide nanoparticles (SPION).⁴⁰ To avoid copper catalysts, strain-promoted azide-alkyne cycloaddition (SPAAC) enables bioorthogonal covalent coupling under milder conditions, successfully coupling dibenzocyclooctyne (DBCO)-modified B-cell lymphoma 2 (Bcl-2) antisense oligonucleotides and hypoxia-inducible factor 1-alpha (HIF-1α) siRNAs with azide-functionalized polylactic acid (PLA)/PEG components.⁶⁷ Covalent click conjugation can also be combined with programmable DNA hybridization. In Ep-SNA@DOX, DBCO-modified Bcl-2 ASOs were first coupled with azide-functionalized PLA through SPAAC, after which complementary trailing DNA hairpins hybridized with the ASOs.⁴²

Multiscale Targeting and in vivo Delivery of SNAs in Cancer

Systemically administered SNAs must navigate a cascade of biological barriers spanning systemic, organ, tumor, cellular, and subcellular levels to achieve therapeutic efficacy. Following intravenous injection, these nanoparticles sequentially encounter serum proteins, physiological clearance mechanisms, tumor tissue stroma, and endolysosomal entrapment. These interconnected processes are collectively governed by SNA size, composition, surface chemistry, oligonucleotide density, targeting ligands, and administration route (Figure 3).

Figure 3.

Multiscale delivery and biological barriers of systemically administered SNAs. Blue boxes indicate successive delivery stages from systemic circulation to intracellular trafficking. Red boxes represent common barriers and off-target pathways encountered by nanoparticles. The Orange box highlights SNA features and design strategies that may help overcome the barriers or reduce off-target delivery indicated by the arrows. Their effects depend on the formulation and biological context. The green box indicates therapeutic activity following successful delivery.

Systemic Fate and Passive Tumor Targeting

In circulation, the highly ordered SNA architecture provides electrostatic shielding that inherently reduces nuclease binding and degradation compared with linear oligonucleotides.⁶⁹ However, serum proteins can adsorb onto the SNA surface and form a protein corona that alters particle size, surface properties, cellular recognition, circulation time, and biodistribution.^70–72 Depending on its composition, this corona can either facilitate cellular uptake or mask targeting ligands, while opsonization generally drives phagocytic clearance and hepatic or splenic accumulation.⁷³

Clearance mechanisms are highly dependent on diverse physicochemical parameters, meaning no universal renal-clearance threshold applies across all SNA formulations. Surface engineering strategies such as PEGylation mitigate nonspecific protein adsorption to prolong circulation.^74,75 Specifically, increasing PEG surface density on RNA-functionalized AuNP-SNAs extended their murine circulation half-life from approximately 1 to 10 minutes.⁷⁶ For LSNAs, utilizing high-affinity diacylglycerol rather than cholesterol anchors extended the serum half-life of surface-bound DNA by over 20-fold while fundamentally altering biodistribution.⁴⁸ Nevertheless, excessive PEG shielding may reduce cellular uptake, and repeated exposure may trigger anti-PEG immune responses.^77,78

Circulating SNAs typically access solid tumors via enhanced permeability and retention (EPR)- associated extravasation, a process influenced by vascular permeability, blood flow, and particle-cell interactions.⁷⁹ Following extravasation, dense extracellular matrices, elevated interstitial fluid pressure, and perivascular binding often restrict deep tissue penetration.⁸⁰ Thus, whole-tumor accumulation does not necessarily indicate uniform tissue distribution or efficient cancer-cell uptake. For instance, tumor accumulation of immunotherapeutic liposome-SNAs is not the only relevant delivery endpoint. Vaccine formulations may need efficient trafficking to draining lymph nodes and uptake by dendritic cells or other antigen-presenting cells rather than maximal accumulation in tumor tissue.⁸¹ Studies modulating lipid composition therefore provide an important conceptual bridge between nanocarrier pharmacokinetics and immunological function: changes in lipid chemistry alter lymph-node accumulation as well as immune activation.⁸² This distinction highlights the differing delivery requirements between cancer vaccines and tumor-targeted gene-regulation systems.

Organ Tropism and Extrahepatic Delivery

The liver and spleen are common sites of nanoparticle accumulation after systemic administration. Liver accumulation may be beneficial for primary liver tumors or hepatic metastases but represents a major barrier to the treatment of extrahepatic tumors. Persistent inorganic cores may also remain in the liver and spleen, raising concerns about long-term safety.²⁶ Therefore, reducing off-target sequestration and improving delivery to other organs remain major challenges for systemically administered SNAs.

Extrahepatic delivery requires SNA designs that address the biological barriers of each organ. Strategies include controlling particle size and surface charge, reducing nonspecific protein adsorption, using biodegradable cores, incorporating tissue- or cell-specific ligands, and selecting local or regional administration routes. For brain tumors, delivery must overcome the blood–brain and blood-tumor barriers. NU-0129 provides preclinical and early clinical evidence that systemically administered AuNP-SNAs can reach glioblastoma tissue.^83,84 Other SNA studies have explored molecular imaging across the blood-brain barrier.⁷⁴ Beyond the brain, ultrasound-responsive SNAs targeting c-Myc and PD-L1 showed increased accumulation in triple-negative breast tumors.⁸⁵ Liposomal SNA composition also influenced organ distribution, with CHOL-LSNAs showing greater DNA accumulation in the lungs and DPPE-LSNAs showing greater accumulation in the kidneys.⁴⁸ But these findings should not be generalized to all SNA formulations.

Active Tumor and Cellular Targeting

Passive tumor accumulation does not ensure selective uptake by cancer cells. Active delivery can be enhanced through tumor-microenvironment-responsive activation or receptor-mediated cellular targeting. These mechanisms are related but distinct: tumor-responsive systems expose or activate SNAs within diseased tissues, whereas targeting ligands promote recognition of receptors enriched on cell populations. Zhang et al⁸⁶ developed MMP-responsive liposomal SNAs coated with zwitterionic polypeptides composed of alternating glutamic acid and lysine residues. The coating reduced nonspecific immune recognition and prolonged circulation. In MMP-rich tumor tissues, the peptide linker was cleaved, and the protective coating was removed, exposing the SNA surface and increasing tumor accumulation and cellular uptake. This example illustrates that responsiveness and targeting can overlap because local activation can simultaneously enhance tissue accumulation and cellular entry. Receptor-mediated targeting can be achieved using antibodies, aptamers, peptides, or small molecules displayed on the SNA surface. Human epidermal growth factor receptor 2 (HER2), which is overexpressed in several malignancies, including subsets of breast, ovarian, and gastric cancer, is an important target for SNA-mediated delivery and gene regulation. Zhang et al⁵¹ demonstrated that anti-HER2 antibody-linked SNAs were taken up more rapidly by HER2-overexpressing SKOV3 ovarian cancer cells than non-targeted SNAs, demonstrating enhanced receptor-dependent uptake.

AS1411 aptamer-functionalized SNAs target nucleolin, which is overexpressed on the surface of several tumor cells, including triple-negative breast cancer cells.⁶⁰ Folate-functionalized SNAs have also been explored to promote uptake by folate-receptor-positive cancer cells.⁸⁷ Overall, combining tumor-responsive activation with receptor-mediated recognition may improve SNA delivery from tumor tissues to specific cancer-cell populations.

Cellular Uptake and Intracellular Trafficking

After reaching the tumor, SNAs must enter target cells and deliver their cargo to the appropriate intracellular compartment. For several AuNP-based SNAs, the oligonucleotide shell is recognized by class A scavenger receptors, enabling efficient cellular uptake without additional transfection reagents. Under specific experimental conditions, AuNP–SNAs can reach approximately 10⁶ particles per cell and are internalized mainly through cholesterol-dependent, lipid raft-associated endocytic pathways, followed by endosomal trafficking.^22,24 However, uptake mechanisms and efficiency may vary among SNA architectures and cell types.

Following internalization, SNAs are generally transported through early and late endosomes and may subsequently accumulate in lysosomes. Therefore, high cellular uptake does not necessarily result in efficient gene regulation. A substantial fraction of the internalized cargo may remain trapped within endolysosomal compartments, where it is either degraded or physically separated from its intracellular target.⁸⁸ Endosomal escape and controlled cargo release are thus critical barriers to SNA-mediated therapy. Strategies such as ionizable lipids, membrane-disruptive peptides, pH-responsive polymers, and enzyme- or reduction-cleavable linkers have been incorporated into SNA systems to destabilize endosomal membranes or release therapeutic oligonucleotides under intracellular conditions.

Intracellular nucleic acid signals can also be used to trigger cargo activation. Zhang et al⁶⁵ demonstrated that cancer-associated miR-21 releases a pistol ribozyme from a DNA-based SNA, enabling the ribozyme to cleave polo-like kinase 1 (PLK1) mRNA. The required intracellular destination depends on the cargo: siRNA and Cas13 primarily act in the cytosol, ASOs may target cytoplasmic or nuclear RNAs, whereas Cas9-based genome-editing components and donor DNA generally require nuclear access.^26,89 Because intact nanoparticle constructs cannot readily undergo passive nuclear diffusion, nuclear activity often depends on active transport or the intracellular release of smaller nucleic acid components.^90,91 For core-retaining SNAs, intracellular cargo release is also critical. Most intact AuNP-SNAs are too large to enter the nucleus efficiently and may remain within endolysosomal vesicles or the cytoplasm. Their activity therefore commonly depends on the release of functional oligonucleotides from the nanoparticle surface through cleavable linkers, intracellular strand displacement, or enzymatic degradation. In comparison, biodegradable lipid, polymeric, protein-based, and self-assembled SNAs can disassemble intracellularly and release smaller nucleic acid components that more readily reach cytosolic or nuclear targets. Nuclear-localization peptides and other organelle-targeting ligands may further improve delivery to the nucleus or specific subcellular sites.

Overall, delivery involves cell recognition, uptake, endosomal escape, cargo release, and transport to the site of action. Improving cellular uptake alone is therefore not enough. SNAs must also protect nucleic acids from degradation and help them reach the correct location inside cells rather than remain trapped in inactive compartments. For systemic treatment, rapid clearance, accumulation in non-target tissues, and toxicity must also be minimized.

Therapeutic Applications of SNAs in Cancer

SNAs have potential for clinical use because they protect oligonucleotides from degradation by nucleases and help them enter cells. Their cellular uptake has been reported to be 2- to 12.5-times that of free oligonucleotides.^52,92 In cancer therapy, SNAs not only function as carriers but also as biologically active nanostructures in which the nucleic acid shell directly mediates gene regulation, genome editing, immune activation, or stimulus-responsive drug release. According to their principal mode of action, therapeutic SNAs can be broadly divided into gene-silencing systems, genome- or transcriptome-editing platforms, cancer immunotherapies, and precision or stimulus-responsive combination treatments.^61,93–95 Although these classes overlap in multifunctional systems, they provide a useful framework for understanding how SNA architecture can be translated into distinct anticancer mechanisms.

Gene Silencing and Oncogene Regulation

Gene silencing is one of the main therapeutic applications of SNAs. Unlike genome editing, siRNAs, ASOs, and miRNAs regulate gene expression without permanently altering genomic DNA. Their activity depends on intracellular localization: siRNAs act in the cytoplasm via RNA interference,⁹⁶ whereas ASOs target cytoplasmic or nuclear RNAs.⁹⁷

siRNA-SNAs effectively suppress anti-apoptotic proteins, oncogenic pathways, and immune checkpoints. AuNP-SNAs against BCL2L12 reduced expression, activated caspases, and inhibited glioblastoma growth in vitro and in vivo,⁸⁴ whereas Gli1-targeting SNAs specifically suppressed key downstream targets, including Cyclin D1, c-Myc, BCL2, and ABCG2, in vitro.⁹⁵ Hairpin-like siRNA-SNAs improve loading, stability, and knockdown duration, achieving persistent suppression of HER2, luciferase, and VEGF in vitro.⁹⁸ Similarly, crosslinked nanogels protecting siRNA during systemic delivery suppressed PLK1 and inhibited MDA-MB-231 tumor growth with an 80% inhibition rate⁹⁹ (Figure 4A). Fe-Shik@Fe-siPD-L1 SNAs achieved a 77.0% reduction in PD-L1 mRNA, suppressing 4T1 tumor growth by 87.4% while mitigating lung metastasis when combined with anti-PD-L1 therapy.⁶⁰

Figure 4.

Representative mechanisms of SNA-mediated gene silencing. (A) SNA-mediated siRNA delivery induces cytoplasmic RNA interference and degradation of target mRNA. Reproduced from Ref. 99. Copyright 2018 Angew Chem Int Ed. (B) SNA-delivered ASOs hybridize with complementary mRNA and promote its degradation. Reproduced from Ref. 94. Copyright 2020 Angew Chem Int Ed. (C) SNA-presented anti-miRNA oligonucleotides bind and inhibit oncogenic miRNAs, thereby restoring downstream gene regulation. Reproduced from Ref. 101. Copyright 2022 Advanced Materials.

ASO-SNAs have been used alone or together with siRNA to regulate oncogenic mRNAs, Long noncoding RNAs (lncRNAs), and microRNAs (miRNAs), including BCL2, HER2, and MALAT1. Light-assisted delivery of BCL2-targeting ASOs promoted intracellular release and suppressed BCL2 expression and HeLa tumor growth⁹⁴ (Figure 4B). Carrier-free SNAs combining HIF-1α siRNA with a BCL2-targeting peptide nucleic acid (PNA)-ASO achieved simultaneous suppression, registering reductions of 81% for HIF-1α and 94% for Bcl-2 alongside an 85% tumor reduction under near-infrared irradiation,⁶¹ while C60-based molecular SNAs containing HER2-targeting oligonucleotides achieved stronger HER2 regulation than corresponding linear sequences in cellular experiments.¹⁰⁰ For nuclear-retained long noncoding RNAs (lncRNAs), phosphorothioate LSNAs targeting lncRNA MALAT1 produced approximately 90% knockdown and increased tumor suppressor interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) expression in vitro.⁹¹ Beyond mRNAs and lncRNAs, ASOs can also inhibit oncogenic miRNAs. Gold-core ASO-SNAs inhibiting miR-21 and miR-155 achieved a 100% survival rate at day 35 in combined gene and photothermal therapy models¹⁰¹ (Figure 4C). More broadly, SNA-mediated miRNA therapy may either restore tumor-suppressive miRNAs or inhibit oncogenic miRNAs. A degradable cyclic-brush SNA delivering miR-122 suppressed hepatocellular carcinoma in vitro and in vivo.¹⁰²

Collectively, SNA architecture enhances stability, intracellular delivery, and activity of gene-silencing oligonucleotides. However, inefficient endosomal escape and limited tumor-selective delivery remain major barriers to clinical translation.

Genome Editing and Emerging Transcriptional Regulation

Genome editing provides permanent gene disruption or correction. Delivering bulky CRISPR components is inherently more complex than siRNA/ASO delivery, requiring precise transport and release into specific intracellular compartments.^103–105 Current SNA research primarily focuses on Cas9. Huang et al¹⁰⁶ developed Cas9-centered ProSNAs preloaded with single-guide RNA, achieving 32–47% in vitro editing efficiency without additional transfection reagents. Recently, CRISPR LNP-SNAs demonstrated 15–68% indel rates (a 2- to 3-fold increase over standard LNPs) and 13–30% homology-directed repair (HDR) efficiencies (up to a 2.5-fold improvement).⁶⁴ Cas9 ribonucleoproteins (RNPs) have also been delivered via related AuNP assemblies for adeno-associated virus integration site 1 (AAVS1) and phosphatase and tensin homolog (PTEN) editing.^107,108 The key steps in SNA-mediated delivery of Cas9 complexes and the subsequent DNA repair pathways are schematically illustrated in Figure 5.

Figure 5.

Representative SNA-mediated CRISPR-Cas9 genome editing. Following cellular uptake, CRISPR-SNAs release Cas9 ribonucleoprotein (RNP) complexes through endosomal escape and cargo release. The complexes enter the nucleus and cleave target DNA. DNA repair through non-homologous end joining (NHEJ) can disrupt target genes, while homology-directed repair (HDR) uses donor DNA for precise editing.

Other CRISPR effectors could broaden the regulatory functions of SNAs. Cas12 may enable DNA editing, whereas Cas13 can achieve programmable RNA degradation without permanently modifying genomic DNA.¹⁰⁹ Nevertheless, most reported combinations of SNAs and Cas12 have been developed for cancer-marker detection rather than therapy.^110,111

Unlike cytosolic siRNA, CRISPR-SNAs need to reach the nucleus to edit genes, which face additional challenges in treating tumors in vivo. Nuclear pore complexes restrict intact constructs >40 nm. CRISPR-SNAs larger than 100 nm cannot easily pass through nuclear pores intact. They may need to break down in the cytoplasm and release their editing cargo, or rely on nuclear envelope breakdown during cell division. This can make editing more difficult in slowly dividing cells.¹¹² Precise gene correction through HDR mainly occurs during the S and G2 phases of the cell cycle. It is therefore inefficient in nondividing tumor cells. NHEJ is active across a broader range of cell cycle phases, but it can introduce insertions or deletions during repair.¹¹³ Pre-existing host immunity to bacterial Cas proteins risks rapid clearance of SNA vehicles or T-cell-mediated destruction of edited cells.¹¹⁴ If Cas remains active for too long, it may also cut DNA at unintended sites, causing unwanted DNA breaks or chromosomal translocations.¹¹⁵ Delivering Cas protein and guide RNA together as an RNP can shorten the duration of editing activity, but efficient loading and stability still need improvement.

Clinical use will require more selective activation of editing systems in tumors and better control over how long they remain active. Treatment efficacy, off-target effects, and safety also need thorough testing in vivo.

Cancer Immunotherapy and Stimulus-Responsive Therapeutics

In contrast to strategies that primarily act through direct regulation of tumor-associated genes, cancer immunotherapy aims to induce or restore host antitumor immune responses. Building on the structural and interfacial properties described above, SNAs can integrate antigens, adjuvants, and immune modulators into a single nanoplatform, enabling applications in cancer vaccines and combination immunotherapy. CpG-containing SNAs activate Toll-like receptor 9 (TLR9) in antigen-presenting cells, with efficacy governed by CpG density, orientation, anchoring chemistry, and release kinetics.^49,116 LSNAs function as active biological nanoparticles, facilitating high-density co-presentation of CpG alongside peptide antigens, neoantigens, or tumor lysates to dictate dendritic cell (DC) activation and cytotoxic T-cell priming.^{50,81,117,118} Multivalent LSNAs bearing MHC-I and MHC-II epitopes simultaneously stimulate cytotoxic and helper T cells^47,81 (Figure 6). Crucially, the structural configuration of these antigens dictates the resulting immune response. For example, N-terminally conjugated, surface-hybridized L-SNAs (N-HSNA) elicited an ~8-fold higher IFN-γ secretion and ~2.5-fold greater CD8+T cell cytotoxicity than simple admixtures, significantly reducing tumor burden in vivo.¹¹⁹ Furthermore, immunostimulatory liposomal SNAs loaded with tumor lysates promoted dendritic-cell activation, increased CD8⁺ T-cell responses, reduced myeloid-derived suppressor cells, and inhibited triple-negative breast cancer in vivo.¹²⁰ Recent work with HPV16 E7/CpG SNA further demonstrated that changing antigen presentation while holding overall composition nearly constant produced large differences in interferon-γ (IFN-γ) production and cytotoxicity, reinforcing the principle that nanoscale geometry is an immunological variable rather than a purely structural one.⁴⁹ For example, substituting cholesterol with a (C12)9 anchor amplified IFN-γ production >200-fold while doubling both memory CD8+ T-cell formation and target cell cytolysis.¹²¹ In parallel, coordination-driven Fe-Gal@Fe-CpG@TF nanovaccines leveraged tumor fragments to promote DC maturation, boost T-cell activation, inhibit orthotopic 4T1 tumors and lung metastasis, and make a long-term anti-metastatic immunological memory.⁶⁰

Figure 6.

Representative Liposomal SNA(LSNAs)-mediated cancer immunotherapy. LSNAs co-deliver antigens and CpG oligonucleotides to dendritic cells. CpG activates Toll-like receptor 9 (TLR9) in endosomes. Antigens are presented through MHC-I and MHC-II to activate CD8⁺ and CD4⁺ T cells, respectively. CD8⁺ T cells kill tumor cells, while CD4⁺ T cells support the CD8⁺ T-cell response.

To overcome immunosuppressive microenvironments, SNAs coordinate immunomodulation with complementary therapies. STAT3 siRNA-carrying CpG-SNAs simultaneously activate TLR9 and suppress immunosuppressive signaling,¹²² whereas doxorubicin- and CpG-loaded LSNAs integrate chemotherapy-induced immunogenic cell death with TLR9 activation,⁶² achieving ~59.9% sustained drug release over 31 days. Nucleic acid nanogels similarly merge PD-L1 silencing with photodynamic immunotherapy.¹²³

What’s more, SNAs undergo structural or functional shifts in response to tumor microenvironments (acidic pH, glutathione (GSH), reactive oxygen species (ROS), hypoxia, enzymes) or external stimuli (light, ultrasound, radiation). Redox- and enzyme-responsive designs, such as Fe-Shik and Fe-Gal SNAs where glutathione and alkaline phosphatase direct on-demand release of 67.7% nucleic acids and 89.5% drugs,⁶⁰ or MMP-responsive LSNAs exhibiting 2- to 3-fold enhanced uptake via protective coating removal,⁸⁶ provide precise spatiotemporal control. External triggers similarly enable combination treatments, including AIE-SNAs and HIF-1α/BCL2 PSNAs coupling gene silencing with photodynamic therapy,^61,94 ultrasound-driven c-Myc/PD-L1 delivery,⁸⁵ and hafnium oxide-based NIR-II imaging and radiotherapy integration⁵³ (Table 2). Overall, while LSNAs excel in co-delivering immunogens and responsive systems enable on-demand tumor activation, clinical translation still faces manufacturing consistency and in vivo safety hurdles.

Table 2.

Representative Stimulus-Responsive SNAs

Design Strategy	Representative System	Therapeutic Method	Trigger	Therapeutic Mechanism	Reference
Redox-responsive	Fe-Shik@Fe-siPD-L1	RNAi	GSH	Programmable release of therapeutic cargos	[60]
Enzyme-responsive	Tumor-associated enzyme-activatable SNA	Gene therapy	MMP	Protective shell removal and SNA activation	[86]
NIR-responsive	PSNA	PDT and dual-gene silencing	NIR	Endosomal escape, HIF-1α/BCL2 suppression, and photodynamic activity	[61]
Ultrasound-responsive	Ultrasound-responsive carrier-free SNA	RNAi	Ultrasound	Ultrasound-triggered on-demand gene release	[85]
Image-guided multifunctional platform	PD-L1-targeted hafnium oxide SNA	RT and Immunotherapy	NIR-II imaging/X-ray	Imaging-guided radiotherapy	[53]

Abbreviations: GSH, glutathione; MMP, matrix metalloproteinase; RNAi, RNA interference; PDT, photodynamic therapy; NIR, near-infrared; RT, radiotherapy; SNA, spherical nucleic acid; NIR-II, second near-infrared window.

Clinical Translation of SNAs: Human Trials and Milestones

Moving from preclinical models to the clinic, early-phase trials have confirmed the bioactivity and safety of SNAs in humans. NU-0129 represents the first-in-human SNA formulation,¹²⁴ evaluated in an early-phase I trial (NCT03020017) for recurrent glioblastoma according to ClinicalTrials. Composed of a 13 nm gold nanoparticle core functionalized with a dense shell of siRNA targeting the oncogene BCL2L12, intravenously administered NU-0129 crossed the intact blood-brain barrier and accumulated within glioma tissue. It triggered target oncoprotein knockdown and tumor cell apoptosis without grade 4/5 treatment-related adverse toxicities. However, metallic cores pose long-term retention and clearance concerns for repetitive clinical use.

To overcome this problem, LSNAs have emerged as the favored platform for cancer immunotherapy. Densely displaying TLR9-agonistic CpG oligonucleotides on a biodegradable lipid vesicle, cavrotolimod (AST-008) advanced into early clinical evaluation for advanced solid malignancies, following an initial Phase 1 study (NCT03086278) demonstrating its safety and immune activation in healthy participants.¹²⁵ Cavrotolimod progressed into a Phase 1b/2 clinical trial (NCT03684785) for advanced solid malignancies, including Merkel cell carcinoma and anti-PD-1-refractory melanoma.¹²⁶ Administered intratumorally alone or in combination with immune checkpoint inhibitors (such as pembrolizumab), cavrotolimod demonstrated immune activation through DC maturation and systemic CD8⁺ T-cell expansion, without heavy metal accumulation risks. Building on these early clinical results, pipelines such as Flashpoint Therapeutics show how SNAs are developing. It includes a CpG-based TLR9 agonist (FLASH-001, in Phase 1/2), vaccines that combine antigens and adjuvants (FLASH-002), and brain-targeted RNA designs for glioblastoma (FLASH-003). These efforts expand the use of SNAs beyond adjuvants to more integrated and targeted cancer treatments.

Together, these clinical findings and emerging programs highlight the potential of LSNAs as a platform for cancer immunotherapy.

Key Challenges and Future Directions

Despite early clinical studies providing essential evidence of biological activity in humans,^83,126 several challenges continue to hinder the clinical translation of SNAs for cancer therapy. Tumor delivery remains inefficient and highly variable because EPR-mediated accumulation is often inconsistent and insufficient.^74,79 In addition, dense stromal barriers, abnormal vasculature, and elevated interstitial fluid pressure can severely restrict nanoparticle penetration, while brain tumor delivery faces further obstacles imposed by the blood-brain and blood-tumor barriers.⁸³

Cellular specificity is also a major concern. Greater tumor accumulation does not guarantee delivery to all tumor cell types. Cancer stem cells and treatment-resistant clones may still escape treatment. In vivo genome editing also faces several challenges such as endosomal entrapment, inefficient nuclear delivery, unintended editing in healthy tissues, and low HDR efficiency (particularly in nondividing tumor cells).^103,127 Furthermore, large-scale manufacturing requires rigorous control of particle size, oligonucleotide density, cargo loading, and batch-to-batch consistency. Long-term safety remains unclear, as inorganic cores and plasmids may remain in the body, Cas proteins may trigger immune responses, and unintended genomic integration or off-target genetic changes may occur. Clinical development is also limited by the lack of standard methods to assess SNA properties, distribution in the body, and long-term safety, as well as clear criteria for regulatory review.

For LSNAs, there are also some problems that need to be solved. After entering cells, how to keep them stable during delivery and release their cargo.²⁰ As for vaccines, it is very important to focus on whether LSNAs could be delivered to lymph nodes and taken up by DC cells rather than tumor accumulation.⁵⁰ What’s more, studies also need to report key formulation properties and performance data consistently so that results can be compared. Effective immune activation should be distinguished from nonspecific inflammation, and systemic toxicity should be assessed. Finally, large-scale production must preserve SNA structure and ensure consistency across batches.

Therefore, future research should focus on biodegradable SNAs with clearly defined molecular components, such as the very promising LSNAs. AI-assisted design and high-throughput screening may help explain how SNA structure affects its activity and guide formulation improvements, so that SNAs could also be designed to reach specific organs, target selected cells, and respond to signals in tumors. However, more complex designs may make cargo loading, release inside cells, and consistent production more difficult. Clear standards should be established to assess their quality, safety, and efficacy.

Conclusions

This review summarizes structures, biological behavior, and cancer applications of SNAs, with a focus on LSNAs for cancer immunotherapy. The effects of SNAs depend on their nucleic acid sequences, core materials, 3D structures, and interactions with the body. Core composition, surface nucleic acid density, and surface chemistry affect their distribution, cellular uptake, immune activation, and treatment outcomes. LSNAs have made progress in gene silencing, cancer immunotherapy, and stimulus-responsive treatments. Some, such as cavrotolimod, have entered clinical trials. Their use in genome editing is still at an early stage, and cell-cycle limitations and safety concerns need further study. A better understanding of how SNA structure affects delivery and treatment outcomes will help guide their development, particularly the use of LSNAs in cancer immunotherapy.

Funding Statement

This research was in part supported by National Institutes of Health grant CA257926 and R01CA303489 to B.Z., and the seed funds from International Institute for Nanotechnology in Northwestern University.

Declaration of AI Use

ChatGPT (GPT-4o, OpenAI) was used to generate illustrative elements with different SNA architectures in Figures 1 and 2. The authors verified their scientific accuracy.

Disclosure

The author(s) report no conflicts of interest in this work.