Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions.

Lipid nanoparticles (LNPs) have become the dominant drug delivery technology in industry, holding the promise to deliver RNA to up or down-regulate any protein of interest. LNPs have mostly been targeted to specific cell types or organs by physicochemical targeting in which LNP's lipid compositions are adjusted to find mixtures with the desired tropism. Here lung-tropic LNPs are examined, whose organ tropism derives from containing either a cationic or ionizable lipid conferring a positive zeta potential. Surprisingly, these LNPs are found to induce massive thrombosis. Such thrombosis is shown in the lungs and other organs, and it is shown that it is greatly exacerbated by pre-existing inflammation. This clotting is induced by a variety of formulations with cationic lipids, including LNPs and non-LNP nanoparticles, and even by lung-tropic ionizable lipids that do not have a permanent cationic charge. The mechanism depends on the LNPs binding to and then changing the conformation of fibrinogen, which then activates platelets and thrombin. Based on these mechanisms, multiple solutions are engineered that enable positively charged LNPs to target the lungs while ameliorating thrombosis. The findings illustrate how physicochemical targeting approaches must be investigated early for risks and re-engineered with a careful understanding of biological mechanisms.

Emerging strategies for nanomedicine in autoimmunity.

Autoimmune disorders have risen to be among the most prevalent chronic diseases across the globe, affecting approximately 5-7% of the population. As autoimmune diseases steadily rise in prevalence, so do the number of potential therapeutic strategies to combat them. In recent years, fundamental research investigating autoimmune pathologies has led to the emergence of several cellular targets that provide new therapeutic opportunities. However, key challenges persist in terms of accessing and specifically combating the dysregulated, self-reactive cells while avoiding systemic immune suppression and other off-target effects. Fortunately, the continued advancement of nanomedicines may provide strategies to address these challenges and bring innovative autoimmunity therapies to the clinic. Through precise engineering and rational design, nanomedicines can possess a variety of physicochemical properties, surface modifications, and cargoes, allowing for specific targeting of therapeutics to pathological cell and organ types. These advances in nanomedicine have been demonstrated in cancer therapies and have the broad potential to advance applications in autoimmunity therapies as well. In this review, we focus on leveraging the power of nanomedicine for prevalent autoimmune disorders throughout the body. We expand on three key areas for the development of autoimmunity therapies - avoiding systemic immunosuppression, balancing interactions with the immune system, and elevating current platforms for delivering complex cargoes - and emphasize how nanomedicine-based strategies can overcome these barriers and enable the development of next-generation, clinically relevant autoimmunity therapies.

Long-term daily oral administration of intestinal permeation enhancers is safe and effective in mice.

Although protein drugs are powerful biologic therapeutics, they cannot be delivered orally because their large size and hydrophilicity limit their absorption across the intestinal epithelium. One potential solution is the incorporation of permeation enhancers into oral protein formulations; however, few have advanced clinically due to toxicity concerns surrounding chronic use. To better understand these concerns, we conducted a 30-day longitudinal study of daily oral permeation enhancer use in mice and resultant effects on intestinal health. Specifically, we investigated three permeation enhancers: sodium caprate (C10), an industry standard, as well as 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC). Over 30 days of treatment, all mice gained weight, and none required removal from the study due to poor health. Furthermore, intestinal permeability did not increase following chronic use. We also quantified the gene expression of four tight junction proteins (claudin 2, claudin 3, ZO-1, and JAM-A). Significant differences in gene expression between untreated and permeation enhancer-treated mice were found, but these varied between treatment groups, with most differences resolving after a 1-week washout period. Immunofluorescence microscopy revealed no observable differences in protein localization or villus architecture between treated and untreated mice. Overall, PPZ and SDC performed comparably to C10, one of the most clinically advanced enhancers, and results suggest that the chronic use of some permeation enhancers may be therapeutically viable from a safety standpoint.

Ionizable lipid nanoparticles deliver mRNA to pancreatic ß cells via macrophage-mediated gene transfer.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing ß cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.

The Effect of Infant Gastric Digestion on Human Maternal Milk Cells.

SCOPE: Human breast milk contains a variety of cell types that have potential roles in infant immunity and development. One challenge associates with defining the purpose(s) of milk cells in the infant is a poor understanding of the effect of digestion on cell fate. METHODS AND RESULTS: This study first demonstrates that milk cell death occurs after gastric digestion in mice. Then flow cytometry and RT-PCR are used to understand the mechanism of human milk cell death and quantify live cell types before and after simulated gastric digestion. This study finds that digestion in simulated gastric fluid for 30 min reduces cell viability from 72% to 27%, with most cell death is caused by the acidic pH. The primary mechanism of cell death is caspase-mediated apoptosis. The non-cellular components of milk offer only mild protection against cell death from stomach acid. CONCLUSIONS: Gastric digestion does not select for a specific resilient cell population to survive-most cell types die in equal proportions in the gastric environment. Taken together, these results provide a foundation with which to understand the fate of human breast milk cells in the infant's intestine and beyond.

The strawberry-derived permeation enhancer pelargonidin enables oral protein delivery.

Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.

Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery.

Therapeutic mRNA has the potential to revolutionize the treatment of myriad diseases and, in 2020, facilitated the most rapid vaccine development in history. Among the substantial advances in mRNA technology made in recent years, the incorporation of base modifications into therapeutic mRNA sequences can reduce immunogenicity and increase translation. However, experiments from our lab and others have shown that the incorporation of base modifications does not always yield superior protein expression. We hypothesized that the variable benefit of base modifications may relate to lipid nanoparticle chemistry, formulation, and accumulation within specific organs. To test this theory, we compared IV-injected lipid nanoparticles formulated with reporter mRNA incorporating five base modifications (ψ, m1ψ, m5U, m5C/ψ, and m5C/s2U) and four ionizable lipids (C12-200, cKK-E12, ZA3-Ep10, and 200Oi10) with tropism for different organs. In general, the m1ψ base modification best enhanced translation, producing up to 15-fold improvements in total protein expression compared to unmodified mRNA. Expression improved most dramatically in the spleen (up to 50-fold) and was attributed to enhanced protein expression in monocytic lineage splenocytes. The extent to which these effects were observed varied with delivery vehicle and correlated with differences in innate immunogenicity. Through comparison of firefly luciferase and erythropoietin mRNA constructs, we also found that mRNA modification-induced enhancements in protein expression are limited outside of the spleen, irrespective of delivery vehicle. These results highlight the complexity of mRNA-loaded lipid nanoparticle drug design and show that the effectiveness of mRNA base modifications depend on the delivery vehicle, the target cells, and the site of endogenous protein expression.

Critical Evaluation of Different Lysosomal Labeling Methods Used to Analyze RNA Nanocarrier Trafficking in Cells.

The use of nucleic acids to regulate gene expression is a rapidly developing field with immense clinical potential. Nanomaterials are frequently used to deliver nucleic acids into cells as they can overcome the poor cellular uptake and endo/lysosomal degradation of bare nucleic acids. For these nanocarriers to be effective, they must escape endo/lysosomal compartments to deliver their nucleic acid cargo into the cytosol (for ribonucleic acid (RNA)) or nucleus (for deoxyribonucleic acid (DNA)). This process is poorly understood and remains an area of active research toward the goal of developing effective delivery strategies. Fluorescent endo/lysosomal markers are among the most widely employed tools used to evaluate the endosomal escape of nucleic acid nanocarriers. However, the endo/lysosomal labeling method may alter the extent of and route of nanocarrier uptake by cells. The impact of these markers on cellular function and cell-nanocarrier interactions has not been probed in a systematic manner. To investigate this, we compared the effects of several common lysosomal labeling methods, namely, LysoTracker Red (LT Red), transient lysosomal-associated membrane protein 1-mutant green fluorescent protein (LAMP1-mGFP) transfection (Transient GFP), and stable lentiviral LAMP1-mGFP transfection (Stable GFP), on cellular metabolic activity, nanocarrier uptake, nanocarrier/lysosomal label colocalization, and gene silencing potency in U87 glioblastoma and MDA-MB-231 breast cancer cells using polyethyleneimine (PEI)/ribonucleic acid (RNA) polyplexes as a model nanocarrier. In both U87s and MDA-MB-231s, Transient GFP and LT Red labeling reduced metabolic activity relative to untransfected (Parental) cells, while Stable GFP labeling increased metabolic activity. Congruently, flow cytometry indicates Stable GFP cells have greater polyplex uptake than LT Red-labeled cells in both cell lines. Despite these similar trends in uptake, polyplex intracellular trafficking differs in the two cell lines, as confocal imaging revealed greater polyplex/lysosome colocalization in Stable GFP U87 cells than LT Red-labeled U87 cells, while the trend was reversed in MBA-MB-231s. The level of RNA-mediated gene silencing achieved in Parental versus Stable GFP U87 and MDA-MB-231 cells agreed with the observed levels of polyplex/lysosome colocalization, supporting the established concept that endosomal escape is the rate-limiting step for RNA interference. These findings indicate that lysosomal labels can profoundly alter cellular function and cell-nanocarrier interactions, presenting critical new considerations for researchers investigating nanoparticle trafficking.

A Potent Branched-Tail Lipid Nanoparticle Enables Multiplexed mRNA Delivery and Gene Editing In Vivo.

The clinical translation of messengerRNA (mRNA) drugs has been slowed by a shortage of delivery vehicles that potently and safely shuttle mRNA into target cells. Here, we describe the properties of a particularly potent branched-tail lipid nanoparticle that delivers mRNA to >80% of three major liver cell types. We characterize mRNA delivery spatially, temporally, and as a function of injection type. Following intravenous delivery, our lipid nanoparticle induced greater protein expression than two benchmark lipids, C12-200 and DLin-MC3-DMA, at an mRNA dose of 0.5 mg/kg. Lipid nanoparticles were sufficiently potent to codeliver three distinct mRNAs (firefly luciferase, mCherry, and erythropoietin) and, separately, Cas9 mRNA and single guide RNA (sgRNA) for proof-of-concept nonviral gene editing in mice. Furthermore, our branched-tail lipid nanoparticle was neither immunogenic nor toxic to the liver. Together, these results demonstrate the unique potential of this lipid material to improve the management of diseases rooted in liver dysfunction.

Layer-by-layer assembled gold nanoshells for the intracellular delivery of miR-34a.

INTRODUCTION: MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation. METHODS: LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-L-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles' ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively. RESULTS: Each successive coating reversed the nanoparticles' charge and increased their hydrodynamic diameter, resulting in a final diameter of 208±4 nm and a zeta potential of 53±5 mV. The LbL-NS released ~30% of their miR-34a cargo over 5 days in 1X PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46±3% and 35±3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects. CONCLUSION: LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.

Spherical Nucleic Acid Architecture Can Improve the Efficacy of Polycation-Mediated siRNA Delivery.

Clinical translation of small interfering RNA (siRNA) nanocarriers is hindered by limited knowledge regarding the parameters that regulate interactions between nanocarriers and biological systems. To address this, we investigated the influence of polycation-based nanocarrier architecture on intracellular siRNA delivery. We compared the cellular interactions of two polycation-based siRNA carriers that have similar size and surface charge but different siRNA orientation: (1) polyethylenimine-coated spherical nucleic acids (PEI-SNAs), in which polyethylenimine is wrapped around a spherical nucleic acid core containing radially oriented siRNA and (2) randomly assembled polyethylenimine-siRNA polyplexes that lack controlled architecture. We found that PEI-SNAs undergo enhanced and more rapid cellular uptake than polyplexes, suggesting a prominent role for architecture in cellular uptake. Confocal microscopy studies demonstrated that while PEI-SNAs and polyplexes exhibit similar intracellular stability, PEI-SNAs undergo decreased accumulation within lysosomes, identifying another advantage conferred by their architecture. Indeed, these advantageous cellular interactions enhanced the gene silencing potency of PEI-SNAs by 10-fold relative to polyplexes. Finally, cytocompatibility studies showed that PEI-SNAs exhibit decreased toxicity per PEI content relative to polyplexes, allowing the use of more polycation. Our studies provide critical insight into design considerations for engineering siRNA carriers and warrant future investigation of how nanocarrier architecture influences cellular-, organ-, and organism-level interactions.

Polyethylenimine-Spherical Nucleic Acid Nanoparticles against Gli1 Reduce the Chemoresistance and Stemness of Glioblastoma Cells.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, with nearly 100% of patients ultimately succumbing to the disease. Median patient survival is 15 months, and no standard of care currently exists for recurrent cases. Glioma stem cells (GSCs), a rare and highly aggressive subpopulation of cells within these tumors, have recently emerged as drivers of tumor initiation and recurrence, and a growing body of evidence suggests that they must be completely eradicated to prevent relapse. Toward this goal, we have developed polyethylenimine-wrapped spherical nucleic acid nanoparticles (PEI-SNAs) targeting Gli1, a transcription factor within the Hedgehog signaling pathway that is crucial for the maintenance of GSCs. Here, we demonstrate that Gli1 PEI-SNAs bind scavenger receptors on GBM cells to undergo endocytosis in a caveolae/lipid raft/dynamin-dependent manner. They further achieve ∼30% silencing of tumor-promoting Hedgehog pathway genes and downstream target genes that promote the aggressive, chemoresistant phenotype of GBM. This produces a 30% decrease in proliferation that correlates with a robust onset of GBM cell senescence as well as an ∼60% decrease in metabolic activity with or without cotreatment with temozolomide (TMZ), the frontline chemotherapy for GBM. Most importantly, Gli1 PEI-SNAs impair the self-renewal capacity of GBM cells as indicated by a 30-40% reduction in the expression of stemness genes and further impair the formation of stem-like neurospheres. They also substantially improve neurosphere chemosensitivity as demonstrated by a 2-fold increase in the fraction of cells undergoing apoptosis in response to low doses of TMZ. These results underscore the potential for siRNA therapeutics targeting Gli1 to reduce GBM resistance to therapy and warrant further development of PEI-SNAs and Gli1-targeted therapies to alleviate drug resistance and recurrence for GBM patients.

Spherical Nucleic Acid Nanoparticles: Therapeutic Potential.

Spherical nucleic acids (SNAs) are highly oriented, well organized, polyvalent structures of nucleic acids conjugated to hollow or solid core nanoparticles. Because they can transfect many tissue and cell types without toxicity, induce minimum immune response, and penetrate various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier), they have become versatile tools for the delivery of nucleic acids, drugs, and proteins for various therapeutic purposes. This article describes the unique structures and properties of SNAs and discusses how these properties enable their application in gene regulation, immunomodulation, and drug and protein delivery. It also summarizes current efforts towards clinical translation of SNAs and provides an expert opinion on remaining challenges to be addressed in the path forward to the clinic.

Enzyme-Linked Immunosorbent Assay to Quantify Targeting Molecules on Nanoparticles.

Molecular targeting presents a promising means of improving the specificity of cancer therapeutics, increasing accumulation at the cancer site and limiting off-target effects. These targeting schemes can be applied to nanoparticle-based treatments to further enhance their anticancer efficacy. Here, we describe methods to conjugate antibodies to silica-gold nanoshells and to quantify the resulting antibody content on the nanoparticles using a solution-based enzyme-linked immunosorbent assay (ELISA). Although we will be using anti-EGFR (epidermal growth factor receptor) antibodies conjugated to gold-silica nanoshells as a model system, this method is adaptable to quantify a range of targeting antibodies and proteins on various types of nanoparticles.

Investigating the role of Hedgehog/GLI1 signaling in glioblastoma cell response to temozolomide.

Resistance to chemotherapy substantially hinders successful glioblastoma (GBM) treatment, contributing to an almost 100% mortality rate. Resistance to the frontline chemotherapy, temozolomide (TMZ), arises from numerous signaling pathways that are deregulated in GBM, including Hedgehog (Hh) signaling. Here, we investigate suppression of Hh signaling as an adjuvant to TMZ using U87-MG and T98G cell lines as in vitro models of GBM. We found that silencing GLI1 with siRNA reduces cell metabolic activity by up to 30% in combination with TMZ and reduces multidrug efflux activity by 2.5-fold. Additionally, pharmacological GLI inhibition modulates nuclear p53 levels and decreases MGMT expression in combination with TMZ. While we surprisingly found that silencing GLI1 does not induce apoptosis in the absence of TMZ co-treatment, we discovered silencing GLI1 without TMZ co-treatment induces senescence as evidenced by a significant 2.3-fold increase in senescence associated ß-galactosidase staining, and this occurs in a loss of PTEN-dependent manner. Finally, we show that GLI inhibition increases apoptosis in glioma stem-like cells by up to 6.8-fold in combination with TMZ, and this reduces the size and number of neurospheres grown from glioma stem-like cells. In aggregate, our data warrant the continued investigation of Hh pathway inhibitors as adjuvants to TMZ chemotherapy and highlight the importance of identifying signaling pathways that determine whether co-treatment will be successful.

Quantification of siRNA Duplexes Bound to Gold Nanoparticle Surfaces.

RNA interference (RNAi)-based gene regulation has recently emerged as a promising strategy to silence genes that drive disease progression. RNAi is typically mediated by small interfering ribonucleic acids (siRNAs), which, upon delivery into the cell cytoplasm, trigger degradation of complementary messenger RNA molecules to halt production of their encoded proteins. While RNAi has enormous clinical potential, its in vivo utility has been hindered because siRNAs are rapidly degraded by nucleases, cannot passively enter cells, and are quickly cleared from the bloodstream. To overcome these delivery barriers, siRNAs can be conjugated to nanoparticles (NPs), which increase their stability and circulation time to enable in vivo gene regulation. Here, we present methods to conjugate siRNA duplexes to NPs with gold surfaces. Further, we describe how to quantify the resultant amount of siRNA sense and antisense strands loaded onto the NPs using a fluorescence-based assay. This method focuses on the attachment of siRNAs to 13 nm gold NPs, but it is adaptable to other types of nucleic acids and nanoparticles as discussed throughout the protocol.

Using Gold Nanoparticles To Disrupt the Tumor Microenvironment: An Emerging Therapeutic Strategy.

Gold nanoparticles have received much attention recently as carriers for anticancer drugs and therapeutic oligonucleotides, but little research has investigated their potential to act as stand-alone therapeutics. Previous studies interrogating their short- and long-term systemic toxicity have found that although gold nanoparticles accumulate within and clear slowly from the liver and spleen, they do not appear to exert toxic effects in these organs. Interestingly, gold nanoparticles innately exhibit the ability to modulate the tumor microenvironment specifically by interfering with crosstalk between tumor cells and stromal cells. In this issue of ACS Nano, Mukherjee and colleagues demonstrate that bare gold nanoparticles can disturb crosstalk between pancreatic stellate cells and pancreatic cancer cells by modulating the cellular secretome to reduce the growth of desmoplastic tissue and inhibit tumor growth. In this Perspective, we highlight opportunities for anticancer targeting within the tumor microenvironment and discuss gold nanoparticles as potential mediators of microenvironment-targeted therapy.

Nanoshell-mediated photothermal therapy can enhance chemotherapy in inflammatory breast cancer cells.

Nanoshell-mediated photothermal therapy (PTT) is currently being investigated as a standalone therapy for the treatment of cancer. The cellular effects of PTT include loss of membrane integrity, so we hypothesized that nanoshell-mediated PTT could potentiate the cytotoxicity of chemotherapy by improving drug accumulation in cancer cells. In this work, we validated our hypothesis using doxorubicin as a model drug and SUM149 inflammatory breast cancer cells as a model cancer subtype. In initial studies, SUM149 cells were exposed to nano-shells and near-infrared light and then stained with ethidium homodimer-1, which is excluded from cells with an intact plasma membrane. The results confirmed that nanoshell-mediated PTT could increase membrane permeability in SUM149 cells. In complementary experiments, SUM149 cells treated with nanoshells, near-infrared light, or a combination of the two to yield low-dose PTT were exposed to fluorescent rhodamine 123. Analyzing rhodamine 123 fluorescence in cells via flow cytometry confirmed that increased membrane permeability caused by PTT could enhance drug accumulation in cells. This was validated using fluorescence microscopy to assess intracellular distribution of doxorubicin. In succeeding experiments, SUM149 cells were exposed to subtherapeutic levels of doxorubicin, low-dose PTT, or a combination of the two treatments to determine whether the additional drug uptake induced by PTT is sufficient to enhance cell death. Analysis revealed minimal loss of viability relative to controls in cells exposed to subtherapeutic levels of doxorubicin, 15% loss of viability in cells exposed to low-dose PTT, and 35% loss of viability in cells exposed to combination therapy. These data indicate that nanoshell-mediated PTT is a viable strategy to potentiate the effects of chemotherapy and warrant further investigation of this approach using other drugs and cancer subtypes.

Elucidating the fundamental mechanisms of cell death triggered by photothermal therapy.

Photothermal therapy (PTT) utilizes nanoparticles embedded within tumors as exogenous energy absorbers to convert laser light energy into heat to ablate cancer cells. While PTT is a promising alternative to conventional cancer therapy, under certain irradiation conditions, it can produce cellular necrosis, and this necrosis may lead to pro-inflammatory responses that are detrimental to treatment success. Recent studies have shown that PTT can be modulated to induce apoptosis rather than necrosis, which is appealing since apoptosis discourages an inflammatory response. In this issue of ACS Nano, del Pino, Pardo, de la Fuente, and colleagues reveal the intracellular signaling cascades involved in the apoptotic response to PTT using cells harboring photothermal transducing nanoprisms. In this Perspective, we present an overview of nanoparticle-mediated PTT and discuss photothermally induced apoptosis as a potential therapeutic pathway.