Excellent Tumor Accumulation Research Articles

Self-assembly of porphyrin-grafted lipid into nanoparticles encapsulating doxorubicin for synergistic chemo-photodynamic therapy and fluorescence imaging.

The limited clinical efficacy of monotherapies in the clinic has urged the development of novel combination platforms. Taking advantage of light-triggered photodynamic treatment combined together with the controlled release of nanomedicine, it has been possible to treat cancer without eliciting any adverse effects. However, the challenges imposed by limited drug loading capacity and complex synthesis process of organic nanoparticles (NPs) have seriously impeded advances in chemo-photodynamic combination therapy. In this experiment, we utilize our previously synthesized porphyrin-grafted lipid (PGL) NPs to load highly effective chemotherapeutic drug, doxorubicin (DOX) for synergistic chemo-photodynamic therapy.Methods: A relatively simple and inexpensive rapid injection method was used to prepare porphyrin-grafted lipid (PGL) NPs. The self-assembled PGL NPs were used further to encapsulate DOX via a pH-gradient loading protocol. The self-assembled liposome-like PGL NPs having a hydrophilic core were optimized to load DOX at an encapsulation efficiency (EE) of ~99%. The resultant PGL-DOX NPs were intact, highly stable and importantly these NPs successfully escaped from the endo-lysosomal compartment after laser irradiation to release DOX in the cytosol. The therapeutic efficacy of the aforementioned formulation was validated both in vitro and in vivo.Results: PGL-DOX NPs demonstrated excellent cellular uptake, chemo-photodynamic response, and fluorescence imaging ability in different cell lines. Under laser irradiation, cells treated with a low molar concentration of PGL-DOX NPs reduced cell viability significantly. Moreover, in vivo experiments conducted in a xenograft mouse model further demonstrated the excellent tumor accumulation capability of PGL-DOX NPs driven by the enhanced permeability and retention (EPR) effect. Through fluorescence imaging, the biodistribution of PGL-DOX NPs in tumor and major organs was also easily monitored in real time in vivo. The inherent ability of porphyrin to generate ROS under laser irradiation combined with the cytotoxic effect of the anticancer drug DOX significantly suppressed tumor growth in vivo.Conclusion: In summary, the PGL-DOX NPs combined chemo-photodynamic nanoplatform may serve as a potential candidate for cancer theranostics.

Implication of multivalent aptamers in DNA and DNA–RNA hybrid structures for efficient drug delivery in vitro and in vivo

To examine proper multivalency and type of aptamers in linear nucleic acid-based structures for efficient intracellular uptakes, we designed linear DNA structures with single and dual aptamers targeting guanosine-rich oligonucleotide 100 (AS1411) and mucin 1 (MUC-1). Multivalent linear DNA structures containing dual types of aptamers showed significantly superior cellular internalization. In addition, multivalent aptamer-immobilized DNA–RNA hybrid structures showed excellent tumor accumulation in vivo without liver toxicity and very low nonspecific cytokine induction. Thus, these multivalent nucleic acid based structures with dual types of aptamers can be specifically applied as carriers of therapeutic nucleic acids without inducing noticeable systemic toxicity.

Molecular Imaging of P-glycoprotein in Chemoresistant Tumors Using a Dual-Modality PET/Fluorescence Probe.

Overexpression of P-glycoprotein (Pgp) has been considered a primary cause for multidrug resistance in a variety of cancers for three decades. However, clinical translation of Pgp targeted therapeutics has been hindered by lack of patient preselection based on the Pgp presence in tumors. We aim to develop a molecularly targeted probe for imaging tumoral Pgp in vivo with positron emission tomography (PET) and fluorescence, and to provide a tool for preselecting the patients with tumoral Pgp expression. Thus, a Pgp monoclonal antibody 15D3 was chemically modified with IRDye800 (IR800) and DOTA chelator. The specificity of the antibody conjugates DOTA-Pab-IR800 was verified in Pgp-expressing 3T3-MDR1 and control 3T3 cells. After radiolabeling with 64Cu, the probe was applied in small animal PET imaging of Pgp in a mouse xenograft model of NCI/ADR-Res cells, which are chemoresistant through overexpression of Pgp. Quantification analysis of the PET images demonstrated that the tumor uptake of the radioactive probe was 9.9 ± 1.4, 12.1 ± 1.2, and 10.5 ± 1.0%ID/g at 4, 24, and 48 h post injection. The tumor-to-muscle ratio was 20.9 at 48 h post injection based on biodistribution studies. Fluorescence imaging was performed following PET experiments, and it demonstrated excellent tumor accumulation of this dual-modality probe in the NCI/ADR-Res tumors. Further, an image-guided surgery was successfully performed using the fluorescence modality of the probe, demonstrating potential utility of this probe in image-guided surgical removal of Pgp-positive drug resistant tumors in the patients. In conclusion, this study clearly demonstrated that the Pgp-targeted antibody probe, 64Cu-DOTA-Pab-IR800, could provide a promising diagnosis tool for detection of Pgp-expressing tumors in vivo.

Plasma membrane activatable polymeric nanotheranostics with self-enhanced light-triggered photosensitizer cellular influx for photodynamic cancer therapy

To address the issue of low cellular uptake of photosensitizers by cancer cells in photodynamic therapy (PDT), we designed a smart plasma membrane-activatable polymeric nanodrug by conjugating the photosensitizer protoporphyrin IX (PpIX) and polyethylene glycol (PEG) with glycol chitosan (GC). The as-prepared GC-PEG-PpIX can self-assemble into core-shell nanoparticles (NPs) in aqueous solution and the fluorescence of PpIX moieties in the inner core is highly quenched due to strong π–π stacking. Interestingly, when encountering plasma membranes, the GC-PEG-PpIX NPs can disassemble and stably attach to plasma membranes due to the membrane affinity of PpIX moieties, which effectively suppresses the self-quenching of PpIX, leading to significantly enhanced fluorescence and singlet oxygen (1O2) production upon laser irradiation. The massively produced 1O2 can compromise the integrity of the plasma membrane, enabling the influx of extracellular nanoagents into cells to promote cell death upon further laser irradiation. Through local injection, the membrane anchored GC-PEG-PpIX enables strong physical association with tumor cells and exhibits highly enhanced in vivo fluorescence at the tumor site. Besides, excellent tumor accumulation and prolonged tumor retention of GC-PEG-PpIX were realized after intravenous injection, which ensured its effective imaging-guided PDT.

Highly Stable Fluorinated Nanocarriers with iRGD for Overcoming the Stability Dilemma and Enhancing Tumor Penetration in an Orthotopic Breast Cancer.

The stability dilemma and limited tumor penetration of nanocarriers in cancer chemotherapy remain two predominant challenges for their successful clinical translation. Herein, the pH-sensitive fluorocarbon-functionalized nanocarriers (SFNs) with a tumor-homing and penetrating peptide iRGD are reported to overcome the stability dilemma and enhance tumor accumulation and penetration in an orthotopic breast cancer. The highly stable SFNs with a low critical association concentration provide a safe and spacious harbor for hydrophobic drugs. Furthermore, the stimulus-responsive evaluation and in vitro drug release study show that the SFNs can balance intracellular dissociation for drug release and extracellular stability in the blood circulation. Additionally, the tumor penetration capacity has been dramatically enhanced in 3D multicellular spheroids, effectively affecting cells far from the periphery. This can be ascribed to the coadministration of iRGD having tumor-penetrating ability and fluorocarbon chains having good cell membrane permeability. The combination of SFNs and iRGD is a viable approach to assist drugs' effective accumulation in primary and metastasized tumor sites, significantly inhibiting the breast tumor growth and curbing lung and liver metastases in an orthotopic-tumor-bearing mouse model. Taken together, this pH-sensitive fluorinated nanosystem having excellent stability and tumor accumulation and penetration properties paves the way to combat cancer.

A next-generation bifunctional photosensitizer with improved water-solubility for photodynamic therapy and diagnosis.

Photodynamic therapy (PDT) exploits light interactions and photosensitizers to induce cytotoxic reactive oxygen species. Photodynamic diagnosis (PDD) uses the phenomenon of photosensitizer emitting fluorescence to distinguish some tumors from normal tissue. The standard photosensitizer used for PDD is 5-aminolevulinic acid (5-ALA), although it is not entirely satisfactory. We previously reported glucose-conjugated chlorin (G-chlorin) as a more effective photosensitizer than another widely used photosensitizer, talaporfin sodium (TS); however, G-chlorin is hydrophobic. We synthesized oligosaccharide-conjugated chlorin (O-chlorin) with improved water-solubility. We report herein on its accumulation and cytotoxicity. O-chlorin was synthesized and examined for solubility. Flow cytometric analysis was performed to evaluate O-chlorin accumulation in cancer cells. To evaluate the intracellular localization of photosensitizer, cells were stained with O-chlorin and organelle-specific fluorescent probes. We then measured the in vitro fluorescence of various photosensitizers and the half-maximal inhibitory concentrations to evaluate effects in PDD and PDT, respectively. Xenograft tumor models were established, and antitumor and visibility effects were analyzed. O-chlorin was first shown to be hydrophilic. Flow cytometry then revealed a 20- to 40-times higher accumulation of O-chlorin in cancer cells than of TS, and a 7- to 23-times greater fluorescence than 5-ALA. In vitro, the cytotoxicity of O-chlorin PDT was stronger than that of TS PDT, and O-chlorin tended to accumulate in lysosomes. In vivo, O-chlorin showed the best effect in PDT and PDD compared to other photosensitizers.O-chlorin was hydrophilic and showed excellent tumor accumulation and fluorescence. O-chlorin is promising as a next-generation bifunctional photosensitizer candidate for both PDT and PDD.

Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon.

For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

Differential tumor-targeting abilities of three single-domain antibody formats

The large molecular size of antibody drugs is considered one major factor preventing them from becoming more efficient therapeutics. Variable regions of heavy chain antibodies (HCAbs), or single-domain antibodies (sdAbs), are ideal building blocks for smaller antibodies due to their molecular size and enhanced stability. In the search for better antibody formats for in vivo imaging and/or therapy of cancer, three types of sdAb-based molecules directed against epidermal growth factor receptor (EGFR) were constructed, characterized and tested. Eleven sdAbs were isolated from a phage display library constructed from the sdAb repertoire of a llama immunized with a variant of EGFR. A pentameric sdAb, or pentabody, V2C-EG2 was constructed by fusing one of the sdAbs, EG2, to a pentamerization protein domain. A chimeric HCAb (cHCAb), EG2-hFc, was constructed by fusing EG2 to the fragment crystallizable (Fc) of human IgG1. Whereas EG2 and V2C-EG2 localized mainly in the kidneys after i.v. injection, EG2-hFc exhibited excellent tumor accumulation, and this was largely attributed to its long serum half life, which is comparable to that of IgGs. The moderate size (∼80 kDa) and intact human Fc make HCAbs a unique antibody format which may outperform whole IgGs as imaging and therapeutic reagents.

Tumor affinity of 99mTc-labeled radiopharmaceuticals, 99mTc-Sn-urokinase and 99mTc-Sn-mannitol

Biolgic distribution of 99mTc-labeled fibrinolytic agent, urokinase, and 99mTc-labeled mannitol, which was obtained as a side-product in the preparation of 99mTc(Sn)-urokinase, have been studied in Ehrlich's tumor-bearing mice to get a promising indicator for the positive delineation of malignant tumor. The preparation of 99mTc-labeled radiopharmaceuticals, 99mTc-UK and 99mTc-Man, was made by the reduction with stannous chloride and labeling efficiency was examined by Sephadex G-25M gel chromatography and by silica gel plate thin layer chromatography. Labeling yield of 99mTc-UK by Sephadex G25M in 0.9% NaCl eluant was 13% and that of 99mTc-Man by TLC in 85% methanol solvent was over 95%. A higher uptake to the implanted solid tumor tissue in mice was found in 99mTc-Man than in 99mTc-UK, of which the excellent tumor accumulation was expected from the positive delineation of malignant tumor with 131I-fibrinogen, 131I-fibrinogen antibody and 125I-plasmin. The poor result in 99mTc-UK, however, may be attributed to the poor fibrinolytic activity of Ehrlich's tumor. In biologic distribution of 99mTc-UK was found high concentration for liver kidney and stomach. In the other hand, a higher tumor tissue uptake, a fast blood disappearance and a low concentration for different organs were found in biologic distribution of 99mTc-Man. Therefore, 99mTc-Man may be assumed as a more preferable 99mTc-labeled tumor localizing radiopharmaceuticals, to which it would be needed as absolute biologic characteristics that 99mTc-labeled compounds possess a high tumor uptake as well as a fast blood disappearance with a low uptake for different organs. However, the possible delineation with 99mTc-labeled fibrinolytic agents, including urokinase and streptokinase, may be promised for malignant tumors in human-subject, which generally have a higher activity in fibrinogenesis than in fibrinolysis.