Targeted Suppression of miRNA-33 Using pHLIP Improves Atherosclerosis Regression.

BACKGROUND: miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. METHODS: We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. RESULTS: The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Col1a2, Fn1, etc) and tissue inhibitor of metalloproteinase 3 (Timp3) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. CONCLUSIONS: This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.

Arginine Residues Modulate the Membrane Interactions of pHLIP Peptides.

Most processes at the water-membrane interface often involve protonation events in proteins or peptides that trigger important biological functions and events. This is the working principle behind the pHLIP peptide technology. A key titrating aspartate (Asp14 in wt) is required to protonate to induce the insertion process, increase its thermodynamic stability when membrane-embedded, and trigger the peptide's overall clinical functionality. At the core of pHLIP properties, the aspartate pKa and protonation are a consequence of the residue side chain sensing the changing surrounding environment. In this work, we characterized how the microenvironment of the key aspartate residue (Asp13 in the investigated pHLIP variants) can be modulated by a simple point mutation of a cationic residue (ArgX) at distinct sequence positions (R10, R14, R15, and R17). We carried out a multidisciplinary study using pHRE simulations and experimental measurements. Fluorescence and circular dichroism measurements were carried out to establish the stability of pHLIP variants in state III and establish the kinetics of the insertion and exit of the peptide from the membrane. We estimated the contribution of the arginine to the local electrostatic microenvironment, which promotes or hinders other electrostatic players from coexisting in the Asp interaction shell. Our data indicate that the stability and kinetics of the peptide insertion and exit from the membrane are altered when Arg is topologically available for a direct salt-bridge formation with Asp13. Hence, the position of arginine contributes to fine-tuning the pH responses of pHLIP peptides, which finds wide applications in clinics.

Kinetics of pHLIP peptide insertion into and exit from a membrane.

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart.

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated "striated muscle contraction" as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.

Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors.

The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including (i) biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; (ii) cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and (iii) tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

Probe for the measurement of cell surface pH in vivo and ex vivo.

We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH-measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7-6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1-6.4, whereas in nonmetastatic tumors, it was 6.7-6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible.

Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo.

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH low insertion peptide (pHLIP) conjugated with a near-infrared fluorescent dye [indocyanine green (ICG)] targets low extracellular pH, allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with nonbuffered saline and instilled with a solution of the ICG pHLIP construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80% if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. The ICG pHLIP imaging agent marked high-grade urothelial carcinomas, both muscle invasive and nonmuscle invasive. Carcinoma in situ was accurately diagnosed in 11 cases, whereas only four cases were seen using white light, so imaging with the ICG pHLIP peptide offers improved early diagnosis of bladder cancers and may also enable new treatment alternatives.

Bilayer Thickness and Curvature Influence Binding and Insertion of a pHLIP Peptide.

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane's hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.

The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions.

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using (31)P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

Enhancement of radiation effect on cancer cells by gold-pHLIP.

Previous research has shown that gold nanoparticles can increase the effectiveness of radiation on cancer cells. Improved radiation effectiveness would allow lower radiation doses given to patients, reducing adverse effects; alternatively, it would provide more cancer killing at current radiation doses. Damage from radiation and gold nanoparticles depends in part on the Auger effect, which is very localized; thus, it is important to place the gold nanoparticles on or in the cancer cells. In this work, we use the pH-sensitive, tumor-targeting agent, pH Low-Insertion Peptide (pHLIP), to tether 1.4-nm gold nanoparticles to cancer cells. We find that the conjugation of pHLIP to gold nanoparticles increases gold uptake in cells compared with gold nanoparticles without pHLIP, with the nanoparticles distributed mostly on the cellular membranes. We further find that gold nanoparticles conjugated to pHLIP produce a statistically significant decrease in cell survival with radiation compared with cells without gold nanoparticles and cells with gold alone. In the context of our previous findings demonstrating efficient pHLIP-mediated delivery of gold nanoparticles to tumors, the obtained results serve as a foundation for further preclinical evaluation of dose enhancement.

Understanding the pharmacological properties of a metabolic PET tracer in prostate cancer.

Generally, solid tumors (>400 mm(3)) are inherently acidic, with more aggressive growth producing greater acidity. If the acidity could be targeted as a biomarker, it would provide a means to gauge the pace of tumor growth and degree of invasiveness, as well as providing a basis for predicting responses to pH-dependent chemotherapies. We have developed a (64)Cu pH (low) insertion peptide (pHLIP) for targeting, imaging, and quantifying acidic tumors by PET, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acid-extruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity, such as hypoxia, lactate dehydrogenase A, and CAIX. We find that the probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.

pH-sensitive pHLIP® coated niosomes.

Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80-90 nm in diameter) using nonionic surfactants Span20 (43-45 mol%), cholesterol (50 mol%) and 5 mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2-3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.

PET Imaging of Extracellular pH in Tumors with (64)Cu- and (18)F-Labeled pHLIP Peptides: A Structure-Activity Optimization Study.

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.

Family of pH (low) insertion peptides for tumor targeting.

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide-lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.

pHLIP peptide targets nanogold particles to tumors.

Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold-pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging.

pH (low) insertion peptide (pHLIP) targets ischemic myocardium.

The pH (low) insertion peptide (pHLIP) family enables targeting of cells in tissues with low extracellular pH. Here, we show that ischemic myocardium is targeted, potentially opening a new route to diagnosis and therapy. The experiments were performed using two murine ischemia models: regional ischemia induced by coronary artery occlusion and global low-flow ischemia in isolated hearts. In both models, pH-sensitive pHLIPs [wild type (WT) and Var7] or WT-pHLIP-coated liposomes bind ischemic but not normal regions of myocardium, whereas pH-insensitive, kVar7, and liposomes coated with PEG showed no preference. pHLIP did not influence either the mechanical or the electrical activity of ischemic myocardium. In contrast to other known targeting strategies, the pHLIP-based binding does not require severe myocardial damage. Thus, pHLIP could be used for delivery of pharmaceutical agents or imaging probes to the myocardial regions undergoing brief restrictions of blood supply that do not induce irreversible changes in myocytes.

Coil-helix transition of polypeptide at water-lipid interface.

We present the exact solution of a microscopic statistical mechanical model for the transformation of a long polypeptide between an unstructured coil conformation and an α-helix conformation. The polypeptide is assumed to be adsorbed to the interface between a polar and a non-polar environment such as realized by water and the lipid bilayer of a membrane. The interfacial coil-helix transformation is the first stage in the folding process of helical membrane proteins. Depending on the values of model parameters, the conformation changes as a crossover, a discontinuous transition, or a continuous transition with helicity in the role of order parameter. Our model is constructed as a system of statistically interacting quasiparticles that are activated from the helix pseudo-vacuum. The particles represent links between adjacent residues in coil conformation that form a self-avoiding random walk in two dimensions. Explicit results are presented for helicity, entropy, heat capacity, and the average numbers and sizes of sboth coil and helix segments.

Targeting breast tumors with pH (low) insertion peptides.

Extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. Thus, targeting of tumor acidity can open new avenues in diagnosis and treatment of aggressive tumors and targeting metastatic cancers cells within a tumor. pH (low) insertion peptides (pHLIPs) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)634Mul transgenic mice with three fluorescently labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs. The Var3- and Var7-pHLIPs constructs have faster blood clearance than the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of the above breast tumor models with tumor accumulation increasing over 4 h postinjection. Staining of nonmalignant stromal tissues in transgenic mice was minimal. The pHLIPs distribution in tumors showed colocalization with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest degree of colocalization of fluorescent pHLIPs was shown to be with lactate dehydrogenase A, which is related to lactate production and acidification of tumors. In sum, the pHLIP-based targeting of breast cancer presents an opportunity to monitor metabolic changes, and to selectively deliver imaging and therapeutic agents to tumors.

Aiming the magic bullet: targeted delivery of imaging and therapeutic agents to solid tumors by pHLIP peptides.

The family of pH (Low) Insertion Peptides (pHLIP) comprises a tumor-agnostic technology that uses the low pH (or high acidity) at the surfaces of cells within the tumor microenvironment (TME) as a targeted biomarker. pHLIPs can be used for extracellular and intracellular delivery of a variety of imaging and therapeutic payloads. Unlike therapeutic delivery targeted to specific receptors on the surfaces of particular cells, pHLIP targets cancer, stromal and some immune cells all at once. Since the TME exhibits complex cellular crosstalk interactions, simultaneous targeting and delivery to different cell types leads to a significant synergistic effect for many agents. pHLIPs can also be positioned on the surfaces of various nanoparticles (NPs) for the targeted intracellular delivery of encapsulated payloads. The pHLIP technology is currently advancing in pre-clinical and clinical applications for tumor imaging and treatment.

Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.

Introduction: We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity.