A specific nanobody-based affinity chromatography resin as a platform for small ubiquitin-related modifier fusion protein purification.

As an excellent fusion tag for expressing heterologous proteins, yeast SUMO (small ubiquitin-related modifier) has unique advantages such as improving solubility, promoting stability, and reducing degradation, but it lacks a simple and rapid purification method. Camelid single-domain antibodies (VHHs or nanobodies) show great promise as an efficient tool in analytical application. In this study, VHHs against SUMO protein were isolated for the first time using biopanning of an immune camelid nanobody library. Among these nanobodies, VS2 demonstrated a high expression level (1.12 g L - 1), and a high affinity for SUMO (2.26 nM). Meanwhile, VHHs were coupled to agarose resins by cysteine at the C-terminal to form affinity chromatography resins. The VS2 resin showed excellent specificity and a dynamic binding capacity for SUMO, SUMO-DsbA (disulfide oxidoreductase) and SUMO-SAM (S-adenosylmethionine synthetase) were 2.41 mg/mL resin, 7.57 mg/mL resin and 16.23 mg/mL resin, respectively. Furthermore, the VS2 resin enabled one-step purification of SUMO-fusions [SUMO-Fc (human IgG1-Fc fragment), SUMO-IGF1 (human insulin-like growth factor 1), SUMO-FGF21 (human fibroblast growth factor 21), SUMO-G-CSF (human Granulocyte colony-stimulating factor), SUMO-PDGF (human platelet-derived growth factor) and SUMO-PAS200 (conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala-and Ser)], and maintained binding capacity and selectivity over 25 purification cycles, each including 15 min of cleaning-in-place with 0.1 M NaOH. This study demonstrated that the VS2 resin was a useful tool at the laboratory scale for one-step purification of various SUMO fusions from complex mixtures.

FAP-Targeted Photodynamic Therapy Mediated by Ferritin Nanoparticles Elicits an Immune Response against Cancer Cells and Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are present in many types of tumors and play a pivotal role in tumor progression and immunosuppression. Fibroblast-activation protein (FAP), which is overexpressed on CAFs, has been indicated as a universal tumor target. However, FAP expression is not restricted to tumors, and systemic treatment against FAP often causes severe side effects. To solve this problem, a photodynamic therapy (PDT) approach was developed based on ZnF16Pc (a photosensitizer)-loaded and FAP-specific single chain variable fragment (scFv)-conjugated apoferritin nanoparticles, or αFAP-Z@FRT. αFAP-Z@FRT PDT efficiently eradicates CAFs in tumors without inducing systemic toxicity. When tested in murine 4T1 models, the PDT treatment elicits anti-cancer immunity, causing suppression of both primary and distant tumors, i.e. abscopal effect. Treatment efficacy is enhanced when αFAP-Z@FRT PDT is used in combination with anti-PD1 antibodies. Interestingly, it is found that the PDT treatment not only elicits a cellular immunity against cancer cells, but also stimulates an anti-CAFs immunity. This is supported by an adoptive cell transfer study, where T cells taken from 4T1-tumor-bearing animals treated with αFAP PDT retard the growth of A549 tumors established on nude mice. Overall, our approach is unique for permitting site-specific eradication of CAFs and inducing a broad spectrum anti-cancer immunity.

NaCl Nanoparticles as a Cancer Therapeutic.

Many inorganic nanoparticles are prepared and their behaviors in living systems are investigated. Yet, common electrolytes such as NaCl are left out of this campaign. The underlying assumption is that electrolyte nanoparticles will quickly dissolve in water and behave similarly as their constituent salts. Herein, this preconception is challenged. The study shows that NaCl nanoparticles (SCNPs) but not salts are highly toxic to cancer cells. This is because SCNPs enter cells through endocytosis, bypassing cell regulations on ion transport. When dissolved inside cancer cells, SCNPs cause a surge of osmolarity and rapid cell lysis. Interestingly, normal cells are much more resistant to the treatment due to their relatively low sodium levels. Unlike conventional chemotherapeutics, SCNPs cause immunogenic cell death or ICD. In vivo studies show that SCNPs not only kill cancer cells, but also boost an anticancer immunity. The discovery opens up a new perspective on nanoparticle-based therapeutics.

Nanoparticle-Laden Macrophages for Tumor-Tropic Drug Delivery.

Macrophages hold great potential in cancer drug delivery because they can sense chemotactic cues and home to tumors with high efficiency. However, it remains a challenge to load large amounts of therapeutics into macrophages without compromising cell functions. This study reports a silica-based drug nanocapsule approach to solve this issue. The nanocapsule consists of a drug-silica complex filling and a solid silica sheath, and it is designed to minimally release drug molecules in the early hours of cell entry. While taken up by macrophages at high rates, the nanocapsules minimally affect cell migration in the first 6-12 h, buying time for macrophages to home to tumors and release drugs in situ. In particular, it is shown that doxorubicin (Dox) as a representative drug can be loaded into macrophages up to 16.6 pg per cell using this approach. When tested in a U87MG xenograft model, intravenously (i.v.) injected Dox-laden macrophages show comparable tumor accumulation as untreated macrophages. Therapy leads to efficient tumor growth suppression, while causing little systematic toxicity. This study suggests a new cell platform for selective drug delivery, which can be readily extended to the treatment of other types of diseases.

Photosensitizer-Encapsulated Ferritins Mediate Photodynamic Therapy against Cancer-Associated Fibroblasts and Improve Tumor Accumulation of Nanoparticles.

Nanoparticles have been widely tested as drug delivery carriers or imaging agents, largely because of their ability to selectively accumulate in tumors through the enhanced permeability and retention (EPR) effect. However, studies show that many tumors afford a less efficient EPR effect and that many nanoparticles are trapped in the perivascular region after extravasation and barely migrate into tumor centers. This is to a large degree attributed to the dense tumor extracellular matrix (ECM), which functions as a physical barrier to prevent efficient nanoparticle extravasation and diffusion. In this study, we report a photodynamic therapy (PDT) approach to enhance tumor uptake of nanoparticles. Briefly, we encapsulate ZnF16Pc, a photosensitizer, into ferritin nanocages, and then conjugate to the surface of the ferritin a single chain viable fragment (scFv) sequence specific to fibroblast activation protein (FAP). FAP is a plasma surface protein widely upregulated in cancer-associated fibroblasts (CAFs), which is a major source of the ECM fiber components. We found that the scFv-conjugated and ZnF16Pc-loaded ferritin nanoparticles (scFv-Z@FRT) can mediate efficient and selective PDT, leading to eradication of CAFs in tumors. When tested in bilateral 4T1 tumor models, we found that the tumor accumulation of serum albumin (BSA), 10 nm quantum dots (QDs), and 50 nm QDs was increased by 2-, 3.5-, and 18-fold after scFv-Z@FRT mediated PDT. Our studies suggest a novel and safe method to enhance the delivery of nanoparticles to tumors.

Protein Nanocage Mediated Fibroblast-Activation Protein Targeted Photoimmunotherapy To Enhance Cytotoxic T Cell Infiltration and Tumor Control.

Carcinoma-associated fibroblasts (CAFs) are found in many types of cancer and play an important role in tumor growth and metastasis. Fibroblast-activation protein (FAP), which is overexpressed on the surface of CAFs, has been proposed as a universal tumor targeting antigen. However, recent studies show that FAP is also expressed on multipotent bone marrow stem cells. A systematic anti-FAP therapy may lead to severe side effects and even death. Hence, there is an urgent need of a therapy that can selectively kill CAFs without causing systemic toxicity. Herein we report a nanoparticle-based photoimmunotherapy (nano-PIT) approach that addresses the need. Specifically, we exploit ferritin, a compact nanoparticle protein cage, as a photosensitizer carrier, and we conjugate to the surface of ferritin a FAP-specific single chain variable fragment (scFv). With photoirradiation, the enabled nano-PIT efficiently eliminates CAFs in tumors but causes little damage to healthy tissues due to the localized nature of the treatment. Interestingly, while not directly killing cancer cells, the nano-PIT caused efficient tumor suppression in tumor-bearing immunocompetent mice. Further investigations found that the nano-PIT led to suppressed C-X-C motif chemokine ligand 12 (CXCL12) secretion and extracellular matrix (ECM) deposition, both of which are regulated by CAFs in untreated tumors and mediate T cell exclusion that prevents physical contact between T cells and cancer cells. By selective killing of CAFs, the nano-PIT reversed the effect, leading to significantly enhanced T cell infiltration, followed by efficient tumor suppression. Our study suggests a new and safe CAF-targeted therapy and a novel strategy to modulate tumor microenvironment (TME) for enhanced immunity against cancer.

Red Blood Cell-Facilitated Photodynamic Therapy for Cancer Treatment.

Photodynamic therapy (PDT) is a promising treatment modality for cancer management. So far, most PDT studies have focused on delivery of photosensitizers to tumors. O2, another essential component of PDT, is not artificially delivered but taken from the biological milieu. However, cancer cells demand a large amount of O2 to sustain their growth and that often leads to low O2 levels in tumors. The PDT process may further potentiate the oxygen deficiency, and in turn, adversely affect the PDT efficiency. In the present study, a new technology called red blood cell (RBC)-facilitated PDT, or RBC-PDT, is introduced that can potentially solve the issue. As the name tells, RBC-PDT harnesses erythrocytes, an O2 transporter, as a carrier for photosensitizers. Because photosensitizers are adjacent to a carry-on O2 source, RBC-PDT can efficiently produce 1O2 even under low oxygen conditions. The treatment also benefits from the long circulation of RBCs, which ensures a high intraluminal concentration of photosensitizers during PDT and hence maximizes damage to tumor blood vessels. When tested in U87MG subcutaneous tumor models, RBC-PDT shows impressive tumor suppression (76.7%) that is attributable to the codelivery of O2 and photosensitizers. Overall, RBC-PDT is expected to find wide applications in modern oncology.

Casein-Coated Fe5C2 Nanoparticles with Superior r2 Relaxivity for Liver-Specific Magnetic Resonance Imaging.

Iron oxide nanoparticles have been extensively used as T2 contrast agents for liver-specific magnetic resonance imaging (MRI). The applications, however, have been limited by their mediocre magnetism and r2 relaxivity. Recent studies show that Fe5C2 nanoparticles can be prepared by high temperature thermal decomposition. The resulting nanoparticles possess strong and air stable magnetism, suggesting their potential as a novel type of T2 contrast agent. To this end, we improve the synthetic and surface modification methods of Fe5C2 nanoparticles, and investigated the impact of size and coating on their performances for liver MRI. Specifically, we prepared 5, 14, and 22 nm Fe5C2 nanoparticles and engineered their surface by: 1) ligand addition with phospholipids, 2) ligand exchange with zwitterion-dopamine-sulfonate (ZDS), and 3) protein adsorption with casein. It was found that the size and surface coating have varied levels of impact on the particles' hydrodynamic size, viability, uptake by macrophages, and r2 relaxivity. Interestingly, while phospholipid- and ZDS-coated Fe5C2 nanoparticles showed comparable r2, the casein coating led to an r2 enhancement by more than 2 fold. In particular, casein coated 22 nm Fe5C2 nanoparticle show a striking r2 of 973 mM(-1)s(-1), which is one of the highest among all of the T2 contrast agents reported to date. Small animal studies confirmed the advantage of Fe5C2 nanoparticles over iron oxide nanoparticles in inducing hypointensities on T2-weighted MR images, and the particles caused little toxicity to the host. The improvements are important for transforming Fe5C2 nanoparticles into a new class of MRI contrast agents. The observations also shed light on protein-based surface modification as a means to modulate contrast ability of magnetic nanoparticles.

Folic acid conjugated ferritins as photosensitizer carriers for photodynamic therapy.

We coupled folic acid as a tumour targeting ligand to the surface of ferritins and loaded them with ZnF16Pc. The resulting nanoconjugates can efficiently hone in on 4T1 tumours in vivo, and, with photoirradiation, leading to suppressed tumour growth and tumour metastasis.

Nanoscintillator-mediated X-ray inducible photodynamic therapy for in vivo cancer treatment.

Photodynamic therapy is a promising treatment method, but its applications are limited by the shallow penetration of visible light. Here, we report a novel X-ray inducible photodynamic therapy (X-PDT) approach that allows PDT to be regulated by X-rays. Upon X-ray irradiation, the integrated nanosystem, comprised of a core of a nanoscintillator and a mesoporous silica coating loaded with photosensitizers, converts X-ray photons to visible photons to activate the photosensitizers and cause efficient tumor shrinkage.

Photostimulable near-infrared persistent luminescent nanoprobes for ultrasensitive and longitudinal deep-tissue bio-imaging.

In vivo fluorescence imaging suffers from suboptimal signal-to-noise ratio and shallow detection depth, which is caused by the strong tissue autofluorescence under constant external excitation and the scattering and absorption of short-wavelength light in tissues. Here we address these limitations by using a novel type of optical nanoprobes, photostimulable LiGa5O8:Cr(3+) near-infrared (NIR) persistent luminescence nanoparticles, which, with very-long-lasting NIR persistent luminescence and unique photo-stimulated persistent luminescence (PSPL) capability, allow optical imaging to be performed in an excitation-free and hence, autofluorescence-free manner. LiGa5O8:Cr(3+) nanoparticles pre-charged by ultraviolet light can be repeatedly (>20 times) stimulated in vivo, even in deep tissues, by short-illumination (~15 seconds) with a white light-emitting-diode flashlight, giving rise to multiple NIR PSPL that expands the tracking window from several hours to more than 10 days. Our studies reveal promising potential of these nanoprobes in cell tracking and tumor targeting, exhibiting exceptional sensitivity and penetration that far exceed those afforded by conventional fluorescence imaging.

Gd-encapsulated carbonaceous dots with efficient renal clearance for magnetic resonance imaging.

Nanoprobes for MRI and optical imaging are demonstrated. Gd@C-dots possess strong fluorescence and can effectively enhance signals on T1 -weighted MR images. The nanoprobes have low toxicity, and, despite a relatively large size, can be efficiently excreted by renal clearance from the host after systemic injection.

Ferritins as nanoplatforms for imaging and drug delivery.

INTRODUCTION: Due to unique architecture and surface properties, ferritin has emerged as an important class of biomaterial. Many studies suggest that ferritin and its derivatives hold great potential in a wide range of bio-applications. AREAS COVERED: In this review, we summarize recent progress on employing ferritins as a platform to construct functional nanoparticles for applications in MRI, optical imaging, cell tracking, and drug delivery. EXPERT OPINION: As a natural polymer, ferritins afford advantages such as high biocompatibility, good biodegradability, and a relatively long plasma half-life. These attributes put ferritins ahead of conventional materials in clinical translation for imaging and drug delivery purposes.

Tumor vasculature targeted photodynamic therapy for enhanced delivery of nanoparticles.

Delivery of nanoparticle drugs to tumors relies heavily on the enhanced permeability and retention (EPR) effect. While many consider the effect to be equally effective on all tumors, it varies drastically among the tumors' origins, stages, and organs, owing much to differences in vessel leakiness. Suboptimal EPR effect represents a major problem in the translation of nanomedicine to the clinic. In the present study, we introduce a photodynamic therapy (PDT)-based EPR enhancement technology. The method uses RGD-modified ferritin (RFRT) as "smart" carriers that site-specifically deliver (1)O2 to the tumor endothelium. The photodynamic stimulus can cause permeabilized tumor vessels that facilitate extravasation of nanoparticles at the sites. The method has proven to be safe, selective, and effective. Increased tumor uptake was observed with a wide range of nanoparticles by as much as 20.08-fold. It is expected that the methodology can find wide applications in the area of nanomedicine.

Improving detection specificity of iron oxide nanoparticles (IONPs) using the SWIFT sequence with long T(2) suppression.

In order to improve the detection specificity of iron oxide nanoparticles (IONPs) delivered to tumors, we embedded saturation pulses into the sweep imaging using Fourier transformation (SWIFT) sequence to suppress long T(2) tissues and fat. Simulation of the Bloch equation was first conducted to study behavior of the saturation pulses of various lengths under different T(2) and off-resonance conditions. MR experiments were then conducted using in vivo mouse xenografts and a phantom consisting of IONPs, vegetable oil, and explanted tumor specimen, without and with long T(2) suppression under a 7T magnetic field. For the in vivo study, arginine-glycine-aspartate (RGD) coated 10nm IONPs (RGD-IONPs) were delivered to tumors implanted in nude mice through both intra-tumor and intravenous injections. Histological studies confirmed that RGD-IONPs efficiently homed to tumors through RGD-integrin interaction. Compared to conventional SWIFT, the proposed method resulted in sufficient suppression on long T(2) species but less influence on short T(2) species. For both the in vivo and ex vivo studies, significantly improved contrast-to-noise ratio (CNR) was achieved between the IONPs and the long T(2) species.

Iron oxide nanoparticle encapsulated diatoms for magnetic delivery of small molecules to tumors.

Small molecules can be co-loaded with iron oxide nanoparticles onto diatoms. With an external magnetic field, the diatoms, after systemic administration, can be attracted to tumors. This study suggests a great potential of diatoms as a novel and powerful therapeutic vehicle.

Fe5C2 nanoparticles with high MRI contrast enhancement for tumor imaging.

An ancient material for magnetic resonance (MR) imaging: For the first time, Fe5C2 is prepared as colloidal stable nanoparticles with good aqueous stability. The nanoparticles boast strong magnetization, excellent chemical inertness, low toxicity, and one of the highest r2 relaxivities reported to date. These nanoparticles hold great potential in MR imaging as well as in other biomedical areas.