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Targeting brain lipid metabolism is a promising strategy to regulate the energy balance and fight metabolic diseases such as obesity. The development of stable platforms for selective delivery of drugs, particularly to the hypothalamus, is a challenge but a possible solution for these metabolic diseases. Attenuating fatty acid oxidation in the hypothalamus via CPT1A inhibition leads to satiety, but this target is difficult to reach in vivo with the current drugs. We propose using an advanced crosslinked polymeric micelle-type nanomedicine that can stably load the CPT1A inhibitor C75-CoA for in vivo control of the energy balance. Central administration of the nanomedicine induced a rapid attenuation of food intake and body weight in mice via regulation of appetite-related neuropeptides and neuronal activation of specific hypothalamic regions driving changes in the liver and adipose tissue. This nanomedicine targeting brain lipid metabolism was successful in the modulation of food intake and peripheral metabolism in mice. info:eu-repo/semantics/publishedVersion

Genome editing using CRISPR/Cas9 has attracted considerable attention for the treatment of genetic disorders and viral infections. Co-delivery of Cas9 mRNA and single guide (sg)RNA is a promising strategy to efficiently edit the genome of various cell types, including non-dividing cells, with minimal safety concerns. However, co-delivery of two RNA species with significantly different sizes, such as Cas9 mRNA (4.5 kb) and sgRNA (0.1 kb), is still challenging, especially in vivo. Here, we addressed this issue by using a PEGylated polyplex micelle (PM) condensing the RNA in its core. PM loading sgRNA alone released sgRNA at minimal dilution in buffer, while PM loading Cas9 mRNA alone was stable even at higher dilutions. Interestingly, co-encapsulating sgRNA with Cas9 mRNA in a single PM prevented sgRNA release upon dilution, which led to the enhanced tolerability of sgRNA against enzymatic degradation. Subsequently, PM with co-encapsulated RNA widely induced genome editing in parenchymal cells in the mouse brain, including neurons, astrocytes, and microglia, following intraparenchymal injection, at higher efficiency than that by co-delivery of PMs loaded with either Cas9 mRNA or sgRNA separately. To the best of our knowledge, this is the first report demonstrating the utility of RNA-based delivery of CRISPR/Cas9 in inducing genome editing in the brain parenchymal cells. Furthermore, the efficiency of genome editing using PMs was higher than using a non-PEGylated polyplex, due to the enhanced diffusion of PMs in the brain tissue. The results reported herein demonstrate the potential of using PMs to co-encapsulate Cas9 mRNA and sgRNA for in vivo genome editing.

Selectively delivering anticancer drugs to solid tumors while avoiding their accumulation in healthy tissues is a major goal in polymeric micelle research. We have recently discovered that the extravasation and permeation of polymeric micelles occur in a dynamic manner characterized by vascular bursts followed by a brief and vigorous outward flow of fluid (called "nano-eruptions"). Nano-eruptions allow delivery of polymeric micelle-associated drugs, though delivery can be heterogeneous both among tumors and within an individual tumor, leading to suboptimal intratumoral distribution. Manipulation of nano-eruptions is expected to improve the efficiency of drug delivery systems (DDSs). By using compounds that affect the intratumoral environment, i.e. a TGF-β inhibitor and chloroquine, the possibility of manipulating nano-eruptions to improve delivery efficiency was investigated. Both compounds were tested in a mouse xenograft model of GFP-labeled pancreatic tumor cells by tracing nano-eruption events and extravasation of size-modulated polymeric micelles in real-time through intravital confocal laser scanning microscopy. The TGF-β inhibitor increased the number of dynamic vents, extended duration time, and generated dynamic vents with a wide range of sizes. Chloroquine did not affect the frequency of nano-eruptions, but it increased tumor vessel diameter, maximum nano-eruption area, and maximum radial increase. Both the TGF-β inhibitor and chloroquine augmented nano-eruptions to diffuse polymeric micelles through tumor stroma, and these medications had a greater effect on the polymeric micelles with larger size, i.e. 70-nm, than on the smaller polymeric micelles having a 30-nm diameter. The results indicate that TGF-β inhibition and chloroquine refashion the intratumoral distribution of DDSs by different mechanisms.

Current strategies to direct therapy-loaded nanoparticles to the brain rely on functionalizing nanoparticles with ligands which bind target proteins associated with the blood-brain barrier (BBB). However, such strategies have significant brain-specificity limitations, as target proteins are not exclusively expressed at the brain microvasculature. Therefore, novel strategies which exploit alternative characteristics of the BBB are required to overcome nonspecific nanoparticle targeting to the periphery, thereby increasing drug efficacy and reducing detrimental peripheral side effects. Here, we present a simple, yet counterintuitive, brain-targeting strategy which exploits the higher impermeability of the BBB to selectively label the brain endothelium. This is achieved by harnessing the lower endocytic rate of brain endothelial cells (a key feature of the high BBB impermeability) to promote selective retention of free, unconjugated protein-binding ligands on the surface of brain endothelial cells compared to peripheral endothelial cells. Nanoparticles capable of efficiently binding to the displayed ligands (i.e., labeled endothelium) are consequently targeted specifically to the brain microvasculature with minimal "off-target" accumulation in peripheral organs. This approach therefore revolutionizes brain-targeting strategies by implementing a two-step targeting method which exploits the physiology of the BBB to generate the required brain specificity for nanoparticle delivery, paving the way to overcome targeting limitations and achieve clinical translation of neurological therapies. In addition, this work demonstrates that protein targets for brain delivery may be identified based not on differential tissue expression, but on differential endocytic rates between the brain and periphery.

Delivering therapeutic antibodies into the brain across the blood-brain barrier at a therapeutic level is a promising while challenging approach in the treatment of neurological disorders. Here, we present a polymeric nanomicelle (PM) system capable of delivering therapeutically effective levels of 3D6 antibody fragments (3D6-Fab) into the brain parenchyma for inhibiting Aβ aggregation. PM assembly was achieved by charge-converting 3D6-Fab through pH-sensitive citraconylation to allow complexation with reductive-sensitive cationic polymers. Brain targeting was achieved by functionalizing the PM surface with glucose molecules to allow interaction with recycling glucose transporter (Glut)-1 proteins. Consequently, 41-fold enhanced 3D6-Fab accumulation in the brain was achieved by using the PM system compared to free 3D6-Fab. Furthermore, therapeutic benefits were obtained by successfully inhibiting Aβ1-42 aggregation in Alzheimer's disease mice systemically treated with 3D6-Fab-loaded glucosylated PM. Hence, this nanocarrier system represents a promising method for effectively delivering functional antibody agents into the brain and treating neurological diseases.

Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood–brain barrier using glycemic control as an external trigger. Glucose‐coated polymeric nanocarriers, which can be bound by glucose transporter‐1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45 nm and an adequate glucose‐ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1 h after intravenous administration and exhibits significant knockdown of a target long non‐coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose‐modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders., The sweet spot: Antisense‐oligonucleotide‐loaded glucosylated‐polyion‐complex micelles can be used for RNA knockdown in various brain regions using blood‐glucose level as an external trigger. Glucose transporter‐1 expressed on the brain capillary endothelial cells binds the glucose‐coated nanocarrier and transports it, along with its cargo, across the blood–brain barrier. This could be used for the treatment of disorders of the central nervous system.

To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly(ethylene glycol)

Prevention of metastatic and local-regional recurrence of cancer after surgery remains difficult. Targeting postsurgical premetastatic niche and microresiduals presents an excellent prospective opportunity but is often challenged by poor therapeutic delivery into minimal residual tumors. Here, an enzymatically transformable polymer-based nanotherapeutic approach is presented that exploits matrix metalloproteinase (MMP) overactivation in tumor-associated tissues to guide the codelivery of colchicine (microtubule-disrupting and anti-inflammatory agent) and marimastat (MMP inhibitor). The dePEGylation of polymersomes catalyzed by MMPs not only exposes the guanidine moiety to improve tissue/cell-targeting/retention to increase bioavailability, but also differentially releases marimastat and colchicine to engage their extracellular (MMPs) and intracellular (microtubules) targets of action, respectively. In primary tumors/overt metastases, the vasculature-specific targeting of nanotherapeutics can function synchronously with the enhanced permeability and retention effect to deter malignant progression of metastatic breast cancer. After the surgical removal of large primary tumors, nanotherapeutic agents are localized in the premetastatic niche and at the site of the postsurgical wound, disrupting the premetastatic microenvironment and eliminating microresiduals, which radically reduces metastatic and local-regional recurrence. The findings suggest that nanotherapeutics can safely widen the therapeutic window to resuscitate colchicine and MMP inhibitors for other inflammatory disorders.

For intravenous delivery of antisense oligonucleotides (ASOs) to solid tumors, a triblock copolymer was synthesized from poly(2-ethyl-2-oxazoline) (PEtOx), poly(2-n-propyl-2-oxazoline) (PnPrOx), an...

Effective inhibition of the protein derived from cellular myelocytomatosis oncogene (c-Myc) is one of the most sought-after goals in cancer therapy. While several c-Myc inhibitors have demonstrated therapeutic potential, inhibiting c-Myc has proven challenging, since c-Myc is essential for normal tissues and tumors may present heterogeneous c-Myc levels demanding contrasting therapeutic strategies. Herein, we developed tumor-targeted nanomedicines capable of treating both tumors with high and low c-Myc levels by adjusting their ability to spatiotemporally control drug action. These nanomedicines loaded homologues of the bromodomain and extraterminal (BET) motif inhibitor JQ1 as epigenetic c-Myc inhibitors through pH-cleavable bonds engineered for fast or slow drug release at intratumoral pH. In tumors with high c-Myc expression, the fast-releasing (FR) nanomedicines suppressed tumor growth more effectively than the slow-releasing (SR) ones, whereas, in the low c-Myc tumors, the efficacy of the nanomedicines was the opposite. By studying the tumor distribution and intratumoral activation of the nanomedicines, we found that, despite SR nanomedicines achieved higher accumulation than the FR counterparts in both c-Myc high and low tumors, the antitumor activity profiles corresponded with the availability of activated drugs inside the tumors. These results indicate the potential of engineered nanomedicines for c-Myc inhibition and spur the idea of precision pH-sensitive nanomedicine based on cancer biomarker levels.

Self-assembled supramolecular structures based on polyion complex (PIC) formation between oppositely charged polymers are attracting much attention for developing drug delivery systems able to endure harsh in vivo environments. As controlling polymer complexation provides an opportunity for engineering the assemblies, an improved understanding of the PIC formation will allow constructing assemblies with enhanced structural and functional capabilities. Here, we focused on the influence of the mixing charge ratio between block aniomers and catiomers on the physicochemical characteristics and in vivo biological performance of the resulting PIC micelles (PIC/m). Our results showed that by changing the mixing charge ratio, the structural state of the core was altered despite the sizes of PIC/m remaining almost the same. These structural variations greatly affected the stability of the PIC/m in the bloodstream after intravenous injection and determined their biodistribution.

Downsizing nanocarriers is a promising strategy for systemically targeting fibrotic cancers, such as pancreatic cancer, owing to enhanced tissue permeability. We recently developed a small oligonucleotide nanocarrier called a unit polyion complex (uPIC) using a single oligonucleotide molecule and one or two molecule(s) of two-branched poly(ethylene glycol)-b-poly( l -lysine) (bPEG-PLys). The uPIC is a dynamic polyion-pair equilibrated with free bPEG-PLys, and thus, is highly stabilized in the presence of excess amounts of free bPEG-PLys in the bloodstream. However, the dynamic polyion-pairing behavior of uPICs needs to be further investigated for longevity in the bloodstream, especially under lower amounts of free bPEG-PLys. Herein, the polyion-pairing behavior of uPICs was investigated by highlighting oligonucleotide stability and negative charge number. To this end, small interfering RNA (siRNA) and antisense oligonucleotides (ASO) were chemically modified to acquire nuclease resistance, and the ASO was hybridized with complementary RNA (cRNA) to form a hetero-duplex oligonucleotide (HDO) with twice the negative charges. While all oligonucleotides similarly formed sub-20 nm-sized uPICs from a single oligonucleotide molecule, the association number of bPEG-PLys (ANbPEG-PLys) in uPICs varied based on the negative charge number of oligonucleotides (N−), that is, ANbPEG-PLys = ~2 at N− = ~40 (i.e., siRNA and HDO) and ANbPEG-PLys = ~1 at N− = 20 (i.e., ASO), presumably because of the balanced charge neutralization between the oligonucleotide and bPEG-PLys with a positive charge number (N+) of ~20. Ultimately, the uPICs prepared from the chemically modified oligonucleotide with higher negative charges showed considerably longer blood retention than those from the control oligonucleotides without chemical modifications or with lower negative charges. The difference in the blood circulation properties of uPICs was more pronounced under lower amounts of free bPEG-PLys. These results demonstrate that the chemical modification and higher negative charge in oligonucleotides facilitated the polyion-pairing between the oligonucleotide and bPEG-PLys under harsh biological conditions, facilitating enhanced blood circulation of uPICs.

Transient in situ stealth coating of liver sinusoids using two-armed PEG relocates nanomedicines from the liver to their targets., A major critical issue in systemically administered nanomedicines is nonspecific clearance by the liver sinusoidal endothelium, causing a substantial decrease in the delivery efficiency of nanomedicines into the target tissues. Here, we addressed this issue by in situ stealth coating of liver sinusoids using linear or two-armed poly(ethylene glycol) (PEG)–conjugated oligo(l-lysine) (OligoLys). PEG-OligoLys selectively attached to liver sinusoids for PEG coating, leaving the endothelium of other tissues uncoated and, thus, accessible to the nanomedicines. Furthermore, OligoLys having a two-armed PEG configuration was ultimately cleared from sinusoidal walls to the bile, while OligoLys with linear PEG persisted in the sinusoidal walls, possibly causing prolonged disturbance of liver physiological functions. Such transient and selective stealth coating of liver sinusoids by two-arm-PEG-OligoLys was effective in preventing the sinusoidal clearance of nonviral and viral gene vectors, representatives of synthetic and nature-derived nanomedicines, respectively, thereby boosting their gene transfection efficiency in the target tissues.

The crucial balance of stability in blood-circulation and tumor-specific delivery has been suggested as one of the challenges for effective bench-to-bedside translation of nanomedicines (NMs). Herein, we developed a supramolecularly enabled tumor-extracellular (Tex) pH-triggered NM that can maintain the micellar structure with the entrapped-drug during systemic circulation and progressively release drug in the tumor by rightly sensing heterogeneous tumor-pH. Desacetylvinblastine hydrazide (DAVBNH), a derivative of potent anticancer drug vinblastine, was conjugated to an aliphatic ketone-functionalized poly(ethylene glycol)-b-poly(amino acid) copolymer and the hydrolytic stability of the derived hydrazone bond was efficiently tailored by exploiting the compartmentalized structure of polymer micelle. We confirmed an effective and safe therapeutic application of Tex pH-sensitive DAVBNH-loaded micelle (Tex-micelle) in orthotopic glioblastoma (GBM) models, extending median survival to 1.4 times in GBM xenograft and 2.6 times in GBM syngeneic model, compared to that of the free DAVBNH. The work presented here offers novel chemical insights into the molecular design of smart NMs correctly sensing Tex-pH via programmed functionalities. The practical engineering strategy based on a clinically relevant NM platform, and the encouraging therapeutic application of Tex-micelle in GBM, one of the most lethal human cancers, thus suggests the potential clinical translation of this system against other types of common cancers, including GBM.

Polyion complex vesicles (PICsomes) formed from a self-assembly of an oppositely charged pair of block- and homo-polyelectrolytes have shown exceptional features for functional loading of bioactive agents. Nevertheless, the stability of PICsomes is often jeopardized in a physiological environment, and only PICsomes having chemically cross-linked membranes have endured in harsh in vivo conditions, such as in the bloodstream. Herein, we developed versatile PICsomes aimed to last in in vivo settings by stabilizing their membrane through a combination of ionic and hydrogen bonding, which is widely found in natural proteins as a salt bridge, by controlled introduction of guanidinium groups in the polycation fraction toward concurrent polyion complexation and hydrogen bonding. The guanidinylated PICsomes were successfully assembled under physiological salt conditions, with precise control of their morphology by tuning the guanidinium content, and the ratio of anionic and cationic components. Guanidinylated PICsomes with 100 nm diameter, which are relevant to nanocarrier development, were stable in high urea concentration, at physiological temperature, and under serum incubation, persisting in blood circulation in vivo.

Polymeric nanoreactors (NRs) have distinct advantages to improve chemical reaction efficiency, but the in vivo applications are limited by lack of tissue-specificity. Herein, novel glucose oxidase (GOD)-loaded therapeutic vesicular NRs (theraNR) are constructed based on a diblock copolymer containing poly(ethylene glycol) (PEG) and copolymerized phenylboronic ester or piperidine-functionalized methacrylate (P(PBEM-co-PEM)). Upon systemic injection, theraNR are inactive in normal tissues. At a tumor site, theraNR are specifically activated by the tumor acidity via improved permeability of the membranes. Hydrogen peroxide (H2O2) production by the catalysis of GOD in theraNR increases tumor oxidative stress significantly. Meanwhile, high levels of H2O2 induce self-destruction of theraNR releasing quinone methide (QM) to deplete glutathione and suppress the antioxidant ability of cancer cells. Finally, theraNR efficiently kill cancer cells and ablate tumors via the synergistic effect.

Structural stability of polyplex micelles (PMs), prepared from plasmid DNA (pDNA) and poly(ethylene glycol)- b -poly( l -lysine) block catiomer (PEG−PLys), was evaluated in terms of their resistance against shear stress. When exposed to shear stress at magnitudes typically present in the blood stream, structural deterioration was observed in PMs owing to the partial removal of PEG−PLys strands. Eventually, impaired PEG coverage of the polyplex core led to accelerated degradation by nucleases, implying that structural deterioration by shear stress in blood stream may be a major cause of rapid clearance of PMs from blood circulation. To address this issue, introduction of disulfide crosslinking into the PM core was shown to be an efficient strategy, which successfully mitigated unfavorable effects of shear stress. Furthermore, improved in vivo blood retention profile and subsequently enhanced antitumor efficacy in systemic treatment of pancreatic adenocarcinoma were confirmed for the crosslinked PMs loaded with pDNA encoding an anti-angiogenic protein, suggesting that high stability under the shear stress during blood circulation may be a critical factor in systemically applicable gene delivery systems.

Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the pH gradient. Subsequent systemic application to the pancreatic tumor demonstrated a capability of vascular targeting mediated by the cRGD ligand, which was directly confirmed based on in situ confocal laser scanning microscopy observation. Encouraging this result, the vascular targeting to transfect a secretable anti-angiogenic gene was attempted to treat the intractable pancreatic tumor with anticipation that the strategy could circumvent the intrinsic physiological barriers derived from hypovascular and fibrotic characters. The obtained therapeutic efficiency demonstrates promising utilities of the proposed formulation as a safe systemic gene delivery carrier in practical use.

This study identifies an oligonucleotide that activates a growth factor receptor and exerts its antiapoptotic activity in vivo., Although growth factors have great therapeutic potential because of their regenerative functions, they often have intrinsic drawbacks, such as low thermal stability and high production cost. Oligonucleotides have recently emerged as promising chemical entities for designing synthetic alternatives to growth factors. However, their applications in vivo have been recognized as a challenge because of their susceptibility to nucleases and limited distribution to a target tissue. Here, we present the first example of oligonucleotide-based growth factor mimetics that exerts therapeutic effects at a target tissue after systemic injection. The aptamer was designed to dimerize a growth factor receptor for its activation and mitigated the progression of Fas-induced fulminant hepatitis in a mouse model. This unprecedented functionality of the aptamer can be reasonably explained by its high nuclease stability and migration to the liver parenchyma. These mechanistic analyses provided insights for the successful application of aptamer-based receptor agonists.

For systemic delivery of small interfering RNA (siRNA) to solid tumors, we developed an actively-targeted unimer polyion complex-assembled gold nanoparticle (uPIC-AuNP) by a two-step assembling process. First is the monodispersed uPIC formation from the single molecules of therapeutic siRNA and the block catiomer, cyclic RGD (cRGD) peptide-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (cRGD-PEG-PLL-LA). Second is the surface decoration of a 20nm-sized AuNP with uPICs. The cRGD-installed uPIC-AuNPs (cRGD-uPIC-AuNP) provided the targetability for selective binding to the cancer and cancer-related endothelial cellular surface, while regulating their size

Enhanced permeability in tumours is thought to result from malformed vascular walls with leaky cell-to-cell junctions. This assertion is backed by studies using electron microscopy and polymer casts that show incomplete pericyte coverage of tumour vessels and the presence of intercellular gaps. However, this gives the impression that tumour permeability is static amid a chaotic tumour environment. Using intravital confocal laser scanning microscopy we show that the permeability of tumour blood vessels includes a dynamic phenomenon characterized by vascular bursts followed by brief vigorous outward flow of fluid (named 'eruptions') into the tumour interstitial space. We propose that 'dynamic vents' form transient openings and closings at these leaky blood vessels. These stochastic eruptions may explain the enhanced extravasation of nanoparticles from the tumour blood vessels, and offer insights into the underlying distribution patterns of an administered drug.

Photodynamic therapy (PDT) is a promising treatment modality for malignant tumors in a light-selective manner. To improve the PDT efficacy, numerous kinds of nanocarriers have been developed to deliver photosensitizers (PSs) selectively into the tumor through leaky tumor-associated vasculature. However, the corresponding prolonged retention of the nanocarrier in the bloodstream may lead to unfavorable photochemical damage to normal tissues such as skin. Here, we report an organic-inorganic hybrid nanocarrier with a pH-responsive on/off switch of PDT efficacy. This hybrid nanocarrier is constructed by hydrothermal synthesis after simple mixing of calcium/phosphate ions, chlorin e6 (amphiphilic low molecular weight PS), and poly(ethylene glycol)-b-poly(aspartic acid) (PEG-PAsp) copolymers in an aqueous solution. The hybrid nanocarrier possesses a calcium phosphate (CaP) core encapsulating the PSs, which is surrounded by a PEG shielding layer. Under physiological conditions (pH 7.4), the nanocarrier suppressed the photochemical activity of PS by lowering the access of oxygen molecules to the incorporated PS, while PDT efficacy was restored in a pH-responsive manner because of the dissolution of CaP and eventual recovery of access between the oxygen and the PS. Owing to this switch, the nanocarrier reduced the photochemical damage in the bloodstream, while it induced effective PDT efficacy inside the tumor cell in response to the acidic conditions of the endo-/lysosomes.

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

International audience; Cancer stem-like cells (CSCs) treatment is a plausible strategy for enhanced cancer therapy. Here we report a glucose-installed sub-50-nm nanocarrier for the targeted delivery of small interfering RNA (siRNA) to CSCs through selective recognition of the glucose ligand to the glucose transporter 1 (GLUT1) overexpressed on the CSC surface. The siRNA nanocarrier was constructed via a two-step assembling process. First, a glucose-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the omega-end (Glu-PEG-PLL-LA) was associated with a single siRNA to form a unimer polyion complex (uPIC). Second, a 20nm gold nanoparticle (AuNP) was decorated with ~65 uPICs through AuS bonding. The glucose-installed targeted nanoparticles (Glu-NPs) exhibited higher cellular uptake of siRNA payloads in a spheroid breast cancer (MBA-MB-231) cell culture compared with glucose-unconjugated control nanoparticles (MeO-NPs). Notably, the Glu-NPs became more efficiently internalized into the CSC fraction, which was defined by aldehyde dehydrogenase (ALDH) activity assay, than the other fractions, probably due to the higher GLUT1 expression level on the CSCs. The Glu-NPs elicited significantly enhanced gene silencing in a CSC-rich orthotopic MDA-MB-231 tumor tissue following systemic administration to tumor-bearing mice. Ultimately, the repeated administrations of polo-like kinase 1 (PLK1) siRNA-loaded Glu-NPs significantly suppressed the growth of orthotopic MDA-MB-231 tumors. These results demonstrate that Glu-NP is a promising nanocarrier design for CSC-targeted cancer treatment.

Antibody fragment (Fab')-installed polyion complex (PIC) micelles were constructed to improve targetability of small interfering RNA (siRNA) delivery to pancreatic cancer cells. To this end, we synthesized a block copolymer of azide-functionalized poly(ethylene glycol) and poly(l-lysine) and prepared PIC micelles with siRNA. Then, a dibenzylcyclooctyne (DBCO)-modified antihuman tissue factor (TF) Fab' was conjugated to azido groups on the micellar surface. A fluorescence correlation spectroscopic analysis revealed that 1, 2, or 3 molecule(s) of Fab'(s) were installed onto one micellar nanoparticle according to the feeding ratio of Fab' (or DBCO) to micelle (or azide). The resulting micelles exhibited ∼40 nm in hydrodynamic diameter, similar to that of the parent micelles before Fab' conjugation. Flow cytometric analysis showed that three molecules of Fab'-installed PIC micelles (3(Fab')-micelles) had the highest binding affinity to cultured pancreatic cancer BxPC3 cells, which are known to overexpress TF on their surface. The 3(Fab')-micelles also exhibited the most efficient gene silencing activity against polo-like kinase 1 mRNA in the cultured cancer cells. Furthermore, the 3(Fab')-micelles exhibited high penetrability and the highest cellular internalization amounts in BxPC3 spheroids compared with one or two molecule(s) of Fab'-installed PIC micelles. These results demonstrate the potential of anti-TF Fab'-installed PIC micelles for active targeting of stroma-rich pancreatic tumors.

The design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid- and polymeric-vesicle-based nanoreactors have some practical limitations. We have succeeded in preparing enzyme-loaded polyion complex vesicles (PICsomes) through a facile protein-loading method. The preservation of enzyme activity was confirmed even after cross-linking of the PICsomes. The cross-linked β-galactosidase-loaded PICsomes (β-gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER-βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4 days after administration of the β-gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme-loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy.

Simultaneous achievement of prolonged retention in blood circulation and efficient gene transfection activity in target tissues has always been a major challenge hindering in vivo applications of nonviral gene vectors via systemic administration. Herein, we constructed novel rod-shaped ternary polyplex micelles (TPMs) via complexation between the mixed block copolymers of poly(ethylene glycol)-b-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) and poly(N-isopropylacrylamide)-b-PAsp(DET) (PNIPAM-b-PAsp(DET)) and plasmid DNA (pDNA) at room temperature, exhibiting distinct temperature-responsive formation of a hydrophobic intermediate layer between PEG shells and pDNA cores through facile temperature increase from room temperature to body temperature (~37 °C). As compared with binary polyplex micelles of PEG-b-PAsp(DET) (BPMs), TPMs were confirmed to condense pDNA into a more compact structure, which achieved enhanced tolerability to nuclease digestion and strong counter polyanion exchange. In vitro gene transfection results demonstrated TPMs exhibiting enhanced gene transfection efficiency due to efficient cellular uptake and endosomal escape. Moreover, in vivo performance evaluation after intravenous injection confirmed that TPMs achieved significantly prolonged blood circulation, high tumor accumulation, and promoted gene expression in tumor tissue. Moreover, TPMs loading therapeutic pDNA encoding an anti-angiogenic protein remarkably suppressed tumor growth following intravenous injection into H22 tumor-bearing mice. These results suggest TPMs with PEG shells and facilely engineered intermediate barrier to inner complexed pDNA have great potentials as systemic nonviral gene vectors for cancer gene therapy.

Recently, nanocarriers that transport bioactive substances to a target site in the body have attracted considerable attention and undergone rapid progression in terms of the state of the art. However, few nanocarriers can enter the brain via a systemic route through the blood-brain barrier (BBB) to efficiently reach neurons. Here we prepare a self-assembled supramolecular nanocarrier with a surface featuring properly configured glucose. The BBB crossing and brain accumulation of this nanocarrier are boosted by the rapid glycaemic increase after fasting and by the putative phenomenon of the highly expressed glucose transporter-1 (GLUT1) in brain capillary endothelial cells migrating from the luminal to the abluminal plasma membrane. The precisely controlled glucose density on the surface of the nanocarrier enables the regulation of its distribution within the brain, and thus is successfully optimized to increase the number of nanocarriers accumulating in neurons., There are only a few examples of nanocarriers that can transport bioactive substances across the blood-brain barrier. Here the authors show that by rapid glycaemic increase the accumulation of a glucosylated nanocarrier in the brain can be controlled.

Protein corona formation was regulated on the surface in vivo by molecular imprinting to enable polymeric nanogels to acquire stealth upon intravenous administration. Albumin, the most abundant protein in blood, was selected as a distinct protein component of protein corona for preparing molecularly imprinted nanogels (MIP-NGs) to form an albumin-rich protein corona. Intravital fluorescence resonance energy transfer imaging of rhodamine-labeled albumin and fluorescein-conjugated MIP-NGs showed that albumin was captured by MIP-NGs immediately after injection, forming an albumin-rich protein corona. MIP-NGs circulated in the blood longer than those of non-albumin-imprinted nanogels, with almost no retention in liver tissue. MIP-NGs also passively accumulated in tumor tissue. These data suggest that this strategy, based on regulation of the protein corona in vivo, may significantly influence the development of drug nanocarriers for cancer therapy.

Nanomedicines capable of smart operation at the targeted site have the potential to achieve the utmost therapeutic benefits. Providing nanomedicines that respond to endogenous stimuli with an additional external trigger may improve the spatiotemporal control of their functions, while avoiding drawbacks from their inherent tissue distribution. Herein, by exploiting the permeabilization of endosomes induced by photosensitizer agents upon light irradiation, we complemented the intracellular action of polymeric micelles incorporating camptothecin (CPT), which can sharply release the loaded drug in response to the reductive conditions of the cytosol, as an effective strategy for precisely controlling the function of these nanomedicines in vivo, while advancing toward a light-activated chemotherapy. These camptothecin-loaded micelles (CPT/m) were stable in the bloodstream, with minimal drug release in extracellular conditions, leading to prolonged blood circulation and high accumulation in xenografts of rat urothelial carcinoma. With the induction of endosomal permeabilization with the clinically approved photosensitizer, Photofrin, the CPT/m escaped from the endocytic vesicles of cancer cells into the cytosol, as confirmed both in vitro and in vivo by real-time confocal laser microscopies, accelerating the drug release from the micelles only in the irradiated tissues. This spatiotemporal switch significantly enhanced the in vivo antitumor efficacy of CPT/m without eliciting any toxicity, even at a dose 10-fold higher than the maximum tolerated dose of free CPT. Our results indicate the potential of reduction-sensitive drug-loaded polymeric micelles for developing safe chemotherapies after activation by remote triggers, such as light, which are capable of permeabilizing endosomal compartments.

The ubiquitin–proteasome system is central in the regulation of cellular proteins controlling cell cycle progression and apoptosis, drawing much interest for developing effective targeted cancer therapies. Herein, we developed a novel pH-responsive polymeric-micelle-based carrier system to effectively deliver the proteasome inhibitor MG132 into cancer cells. MG132 is covalently bound to the block copolymer composed of polyethylene glycol (PEG) and polyaspartate through an acid-labile hydrazone bond. This bond is stable at physiological condition, but hydrolytically degradable in acidic compartments in the cell, such as late-endosomes and lysosomes, and thus, it was used for controlled release of MG132 after EPR-mediated preferential accumulation of the micelles into the tumor. MG132-loaded micelles have monodispersed size distribution with an average diameter of 45 nm, and critical micelle concentration is well below 10 − 7 M. In vitro studies against several cancer cell lines confirmed that MG132-loaded micelles retained the cytotoxic effect, and this activity was indeed due to the inhibition of proteasome by released MG132 from the micelles. Real-time in vitro confocal-microscopy experiments clearly indicated that MG132-conjugated micelles disintegrated only inside the target cells. By intravital confocal micro-videography, we also confirmed the prolonged circulation of MG132 loaded micelles in the bloodstream, which lead to tumor specific accumulation of micelles, as confirmed by in vivo imaging 24 h after injection. These micelles showed significantly lower in vivo toxicity than free MG132, while achieving remarkable antitumor effect against a subcutaneous HeLa-luc tumor model. Our findings create a paradigm for future development of polymeric-micelle-based carrier system for other peptide aldehyde type proteasome inhibitors to make them effective cohort of the existing cancer therapeutic regiments.

For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong potential of uPIC-AuNP nanoarchitectures for systemic siRNA delivery to solid tumors.

Stabilisation of fragile oligonucleotides, typically small interfering RNA (siRNA), is one of the most critical issues for oligonucleotide therapeutics. Many previous studies encapsulated oligonucleotides into ~100-nm nanoparticles. However, such nanoparticles inevitably accumulate in liver and spleen. Further, some intractable cancers, e.g., tumours in pancreas and brain, have inherent barrier characteristics preventing the penetration of such nanoparticles into tumour microenvironments. Herein, we report an alternative approach to cancer-targeted oligonucleotide delivery using a Y-shaped block catiomer (YBC) with precisely regulated chain length. Notably, the number of positive charges in YBC is adjusted to match that of negative charges in each oligonucleotide strand (i.e., 20). The YBC rendezvouses with a single oligonucleotide in the bloodstream to generate a dynamic ion-pair, termed unit polyion complex (uPIC). Owing to both significant longevity in the bloodstream and appreciably small size (~18 nm), the uPIC efficiently delivers oligonucleotides into pancreatic tumour and brain tumour models, exerting significant antitumour activity., Nanoparticle delivery of siRNA has problems with penetration and off target accumulation. Here, the authors report on the development of Y-shaped block catiomers which dynamically wrap around siRNA; demonstrate increased circulation times and delivery into hard to reach brain and pancreas tumour models.

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting αvβ3/αvβ5 integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, "cyclic-Arg-Ala-Asp" (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred via an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma.

For systemic small interfering RNA (siRNA) delivery to tumor mass, a multifunctional polyion complex micelle was constructed with a block copolymer bearing a targeting ligand and a micelle-stabilizing moiety as well as an endosome-disrupting cationic unit. The block copolymer was comprised of poly(ethylene glycol) (PEG) and a polyaspartamide derivative with flanking cationic tetraethylenepentamine (TEP) moiety (PAsp(TEP)), in which the distal ends of PEG and PAsp(TEP) were further installed with cyclic RGD (cRGD) peptide ligand and cholesteryl (Chol) moiety, respectively. The resulting polymer was confirmed to form siRNA-loaded micelle with a diameter of sub 50 nm and a narrow size distribution. In the stability assays with fluorescently labeled siRNA, the terminal Chol moiety significantly suppressed both the rapid dissociation of the micelles in the serum-containing medium and their rapid elimination from the bloodstream, presumably due to its hydrophobic interactions in the micellar core. Moreover, the targeting cRGD ligand, associated with the stabilizing moiety, significantly enhanced the accumulation of siRNA-loaded micelle in a subcutaneous lung (A549) tumor, compared to a non-targeted control, after systemic administration. Ultimately, significant tumor growth inhibition was achieved by systemic administration of the targeted/stabilized micelle incorporating polo-like kinase 1 (Plk1) siRNA with negligible liver toxicity, consistent with the significant sequence-specific gene silencing of Plk1 in the tumor tissue. These results demonstrated the therapeutic potential of cRGD-PEG-PAsp(TEP)-Chol/siRNA micelle for systemic siRNA delivery toward cancer therapy.

Background: Excessive accumulation of β-amyloid (Aβ) has been proposed as a pivotal event in the pathogenesis of Alzheimer’s disease. Possible mechanisms underlying Aβ-induced neuronal cytotoxicity include excess production of reactive oxygen species (ROS) and apoptosis. We have designed novel nanoparticles, nitroxyl radical-containing nanoparticles (RNPs), which possess nitroxyl radical in the core and chemically scavenges ROS. This study aimed to determine the potential neuroprotective role of RNPs on Aβ-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Method: SH-SY5Y cells were preincubated with 0.1–1 mM RNP for 24 h and then incubated with 20 µM Aβ1–42 for 48 h. In every group, cell viability, apoptotic rate, ROS levels including superoxide anion radicals and hydroxyl radicals, ROS production including lipid peroxidation, protein oxidation and DNA oxidation were measured. Results: SH-SY5Y cells preincubated with 0.1–2 mM RNP for 24 h were protected from Aβ-induced damage. SH-SY5Y cells preincubated with more than 2 mM RNP for 24 h showed cytotoxicity. From the quantitative analyses, it was observed that RNPs reduced intracellular oxidative stress. RNP treatment significantly reduced the amount of oxidized lipids, proteins and DNA. It also reduced DNA fragmentations, which caused lower apoptosis levels. Conclusion: RNPs are promising intracellular ROS scavengers.

The receptor activator of NFκB ligand (RANKL) induces Ca(2+) oscillations and activates the Nuclear Factor of Activated T cells 1 (NFATc1) during osteoclast differentiation (osteoclastogenesis). Ca(2+) oscillations are an important trigger signal for osteoclastogenesis, however the molecular basis of Ca(2+) permeable influx pathways serving Ca(2+) oscillations has not yet been identified. Using a DNA microarray, we found that Transient Receptor Potential Vanilloid channels 2 (TRPV2) are expressed significantly in RANKL-treated RAW264.7 cells (preosteoclasts) compared to untreated cells. Therefore, we further investigated the expression and functional role of TRPV2 on Ca(2+) oscillations and osteoclastogenesis. We found that RANKL dominantly up-regulates TRPV2 expression in preosteoclasts, and evokes spontaneous Ca(2+) oscillations and a transient inward cation current in a time-dependent manner. TRPV inhibitor ruthenium red and tetracycline-induced TRPV2 silencing significantly decreased both the frequency of Ca(2+) oscillations and the transient inward currents in RANKL-treated preosteoclasts. Silencing of store-operated Ca(2+) entry (SOCE) proteins similarly suppressed both RANKL-induced oscillations and currents in preosteoclasts. Furthermore, suppression of TRPV2 also reduced RANKL-induced NAFTc1 expression, its nuclear translocation, and osteoclastogenesis. In summary, Ca(2+) oscillations in preosteoclasts are triggered by RANKL-dependent TRPV2 and SOCE activation and intracellular Ca(2+) release. Subsequent activation of NFATc1 promotes osteoclastogenesis.

The antioxidant capacity of lignin from used green tea leaves was evaluated by measuring its effects on the autoxidation of linoleic acid. Autoxidation of linoleic acid was reduced by 50% in the presence of tea leaf lignin. This effect was weaker than that of α-tocopherol and t-butylhydroxyanisole. However, lignin enhanced the antioxidant effects of α-tocopherol, epigallocatechin gallate or acetone extracts of used tea leaves, with the antioxidant activity of lignin + acetone extracts of used tea leaves being nearly equivalent to that of α-tocopherol and t-butylhydroxyanisole. PRACTICAL APPLICATIONS The agricultural industry generates large amounts of botanical food by-products, including used tea leaves, most of which are discarded. To determine whether lignin extracted from used green tea leaves has a practical application, we measured its effect on the autoxidation of linoleic acid, a model for the oxidation of fats. We found that lignin contained in used green tea leaves has an antioxidant effect, suggesting that used tea leaves in industrial waste may have potential applications as antioxidants for food oils and fats.

ClC-7 Cl− channels expressed in osteoclasts are important for bone resorption since it has been shown that disruption of the ClCN7 gene in mice leads to severe osteopetrosis. We have previously reported that Cl− currents recorded from mouse osteoclasts resemble those of ClC-3 Cl− channels. The aim of the present study was to determine the expression of ClC-3 channels in mouse osteoclasts and their functional role during bone resorption. We detected transcripts for both ClC-7 and ClC-3 channels in mouse osteoclasts by RT-PCR. The expression of ClC-3 was confirmed by immunocytochemical staining. Mouse osteoclasts lacking ClC-3 Cl− channels (ClC-3−/− osteoclasts) derived from ClCN3 gene-deficient mice (ClC-3−/−) showed lower bone resorption activity compared with ClC-3+/+ osteoclasts derived from wild-type mice (ClC-3+/+). Treatment of ClC-3+/+ osteoclasts with small interfering RNA (siRNA) against ClC-3 also significantly reduced bone resorption activity. Electrophysiological properties of basal and hypotonicity-induced Cl− currents in ClC-3−/− osteoclasts did not differ significantly from those in ClC-3+/+ osteoclasts. Using immunocytochemistry, ClC-3 was colocalized with lysosome-associated membrane protein 2. Using pH-sensitive dyes, organelle acidification activity in ClC-3−/− osteoclasts was weaker than in ClC-3+/+ osteoclasts. Treatment of ClC-3+/+ osteoclasts with siRNA against ClC-3 also reduced the organelle acidification activity. In conclusion, ClC-3 Cl− channels are expressed in intracellular organelles of mouse osteoclasts and contribute to osteoclastic bone resorption in vitro through organelle acidification.

Human papillomavirus (HPV) E6 and E7 oncogenes are essential for the immortalization and maintenance of HPV-associated cancer and are ubiquitously expressed in cervical cancer lesions. Small interfering RNA (siRNA) coding for E6 and E7 oncogenes is a promising approach for precise treatment of cervical cancer, yet a delivery system is required for systemic delivery to solid tumors. Here, an actively targeted polyion complex (PIC) micelle was applied to deliver siRNAs coding for HPV E6/E7 to HPV cervical cancer cell tumors in immune-incompetent tumor-bearing mice. A cell viability assay revealed that both HPV type 16 and 18 E6/E7 siRNAs (si16E6/E7 and si18E6/E7, respectively) interfered with proliferation of cervical cancer cell lines in an HPV type-specific manner. A fluorescence imaging biodistribution analysis further revealed that fluorescence dye-labeled siRNA-loaded PIC micelles efficiently accumulated within the tumor mass after systemic administration. Ultimately, intravenous injection of si16E6/E7 and si18E6/E7-loaded PIC micelles was found to significantly suppress the growth of subcutaneous SiHa and HeLa tumors, respectively. The specific activity of siRNA treatment was confirmed by the observation that p53 protein expression was restored in the tumors excised from the mice treated with si16E6/E7- and si18E6/E7-loaded PIC micelles for SiHa and HeLa tumors, respectively. Therefore, the actively targeted PIC micelle incorporating HPV E6/E7-coding siRNAs demonstrated its therapeutic potential against HPV-associated cancer.

A dose circulating through the blood at one time will have different opportunities to access the tumor compared to a dose circulating hours later. Methods to test this hypothesis allowed us to differentiate two uniquely fluorescent doses of nanoparticles (administered as a mixture or sequentially) and to measure the distribution and correlation of these nanoparticle doses in three dimensions. Multiple colocalization analyses confirm that silica nanoparticles separated into different dose administrations will not accumulate in the same location. Decreased colocalization between separate doses implies dynamic extravasation events on the scale of microns. Further, the perfusion state of different blood vessels can change across the dosing period. Lastly, analyzing the distance traveled by these silica nanoparticles in two dimensions can be an overestimation when compared with three-dimensional distance analysis. Better understanding intratumoral distribution of delivered drugs will be crucial to overcoming the various barriers to transport in solid tumors.

Occult nodal metastases increase the risk of cancer recurrence, demoting prognosis and quality of life of patients. While targeted drug delivery by using systemically administered nanocarriers can potentially control metastatic disease, lymph node metastases have been mainly dealt by locally injecting nanocarriers, which may not always be applicable. Herein, we demonstrated that sub-50 nm polymeric micelles incorporating platinum anticancer drugs could target lymph node metastases in a syngeneic melanoma model after systemic injection, even after removing the primary tumors, limiting the growth of the metastases. By comparing these micelles with clinically used doxorubicin-loaded liposomes (Doxil) having 80 nm, as well as a 70 nm version of the micelles, we found that the targeting efficiency of the nanocarriers against lymph node metastases was associated with their size-regulated abilities to extravasate from the blood vasculature in metastases and to penetrate within the metastatic mass. These findings indicate the potential of sub-50 nm polymeric micelles for developing effective conservative treatments against lymph node metastasis capable of reducing relapse and improving survival.