A Comparative Study of ^(99m)Tc-YIGSR and 99mTc-MIBI Uptake in Tumor Cells

To investigate a new kind of tumor tracer ^99mTc-YIGSR developed from a five amino structure （YIGSR） of the Laminin -chain, which can bind to the laminin receptors of tumor specifically, and radiolabeled with MAG3. （1） Preparation of the ^99mTc-YIGSR probe： with S-Acetly-NH3- MAG3as the chelator and with proper reductants YIGSR was labeled with ^99mTc;（2） Cell culture and viability measurement： EAC was maintained in RPMI 1640 supplemented with calf serum; the trypan blue exclusion was applied to calculate the cell viability; （3） Study of the cell dynamic： The EAC＇s uptake of ^99mTc-YIGSR and ^99mTc-MIBI was observed at 37℃ and 22 ℃, respectively. （1） The labeling efficiencies of ^99mTc-YIGSR and^99mTc-MIBI were （62±3） % and （96±2） %, respectively; （2） The cell viability was declined with time of incubation; （3） At 37 ℃, the EAC＇S uptake of ^99mTc YIGSR and^99mTc-MIBI reached the peak of （43. 16±2.4） % and （24.4±1.8） % at 60 min, respectively; and at 22 ℃, the highest uptake was （26.5±2. 1） % and （9.47±1.9） % at 60 min, respectively. The in vitro study suggests that ^99mTc-YIGSR is superior to ^99mTc-MIBI in cell uptake and has potential value in tumor imaging.

The impact of Rho GTPases on the cell uptake of single-walled carbon nanohorns

Rho GTPases play an important role on the regulation of cytoskeleton, which can affect the cell morphogenesis, cell migration, endocytosis and vesicle transport by controlling the growth and maintenance of microfilaments and microtubules. It has been known that regulation of cell cytoskeleton is inseparable from the cell uptake of nano-medicine or nano-drug delivery systems. However, only few studies have focused on the impacts of Rho GTPases on cell uptake of nano-medicine or nano-drug delivery systems. This study selected single-walled carbon nanohoms （SWCNHs）, which have emerged as promising drug delivery systems, to explore the impacts of Rho GTPases on cell uptake of nano-drug delivery systems. SWCNHs were oxidized with concentrated nitric acid and prepared into nano dispersion by ultrasonic dispersion. Confocal laser scanning microscope （CLSM） and transmission electron microscopy （TEM） were used to observe the cell uptake and intracellular distribution of nanoparticles after incubated A549 cells with the dispersion mentioned above. Mechanism of cell uptake was assessed using various inhibitors. The results showed that the cell uptake of oxSWCNHs was significantly reduced when RhoA was inhibited. The oxSWCNHs were internalized through clathrin-mediated endocytosis and mainly positioned in lysosomes ofA549 cells.

Fabrication of self-assembling nanofibers with optimal cell uptake and therapeutic delivery efficacy

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy.In this contribution,we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides(MDPs)and use them as a model system to investigate structure-dependent cell penetrating activity.MDPs selfassembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane.However,despite the multivalent charge presentation,not all fibers led to high levels of membrane activity and cellular uptake.The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation.Nanofibers were found to sacrifice their dimension,thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction.The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation.In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone.Through these detailed structure and activity studies,the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.

Uptake of oxidative stress-mediated extracellular vesicles by vascular endothelial cells under low magnitude shear stress

Extracellular vesicles(EVs)are increasingly used as delivery vehicles for drugs and bioactive molecules,which usually require intravascular administration.The endothelial cells covering the inner surface of blood vessels are susceptible to the shear stress of blood flow.Few studies demonstrate the interplay of red blood cell-derived EVs(RBCEVs)and endothelial cells.Thus,the phagocytosis of EVs by vascular endothelial cells during blood flow needs to be elucidated.In this study,red blood cell-derived extracellular vesicles(RBCEVs)were constructed to investigate endothelial cell phagocytosis in vitro and animal models.Results showed that low magnitude shear stress including low shear stress(LSS)and oscillatory shear stress(OSS)could promote the uptake of RBCEVs by endothelial cells in vitro.In addition,in zebrafish and mouse models,RBCEVs tend to be internalized by endothelial cells under LSS or OSS.Moreover,RBCEVs are easily engulfed by endothelial cells in atherosclerotic plaques exposed to LSS or OSS.In terms of mechanism,oxidative stress induced by LSS is part of the reason for the increased uptake of endothelial cells.Overall,this study shows that vascular endothelial cells can easily engulf EVs in areas of low magnitude shear stress,which will provide a theoretical basis for the development and utilization of EVs-based nano-drug delivery systems in vivo.

Personalized cancer vaccines from bacteria-derived outer membrane vesicles with antibody-mediated persistent uptake by dendritic cells

Nanocarriers with intrinsic immune adjuvant properties can activate the innate immune system while delivering tumor antigen,thus efficiently facilitating antitumor adaptive immunity.Bacteria-derived outer membrane vesicles(OMVs)are an excellent candidate due to their abundance of pathogen associated molecular patterns.However,during the uptake of OMVs by dendritic cells(DCs),the interaction between lipopolysaccharide and toll-like receptor 4 induces rapid DC maturation and uptake blockage,a phenomenon we refer to as“maturation-induced uptake obstruction"(MUO).Herein we decorated OMV with the DC-targeting aDEC205 antibody(OMV-DEC),which endowed the nanovaccine with an uptake mechanism termed as 4<not restricted to maturation via antibody modifying”(Normandy),thereby overcoming the MUO phenomenon.We also proved the applicability of this nanovaccine in identifying the human tumor neoantigens through rapid antigen display.In summary,this engineered OMV represents a powerful nanocarrier for personalized cancer vaccines,and this antibody modification strategy provides a reference to remodel the DC uptake pattern in nanocarrier design.

Surface-ligand effect on radiosensitization of ultrasmall luminescent gold nanoparticles

Gold nanoparticles(AuNPs)could serve as pot ential radiother apy sensitizers because of their exceptional biocompatibility and high.Z material nature;however,since in vitro and in vivo behaviors of AuNPs are determined not only by their particle size but also by their surface chemistries,whether surface ligands can affect their radiosensitization has seldom been investi-gated in the radiosensitization of AuNPs.By conducting head-to-head comparison on radio-sensitization of two kinds of ultrasmall(~2 nm)near-infrared(NIR)emitting AuNPs that are coated with zwitterionic glutathione and neutr al polyethylene glyol(PEG)ligands,respectively,we found that zwitterionic glut athione coated AuNPs(GS-AuNPs)can reduce survival rates of MCF-7 cells under irr adiation of clinically used megavoltage photon beam at low dosage of~2.25 Gy.On the other hand,PEG-AuNPs can serve as a radiation-protecting agent and enabled MCF-7 cells more resistant to the irradiation,clearly indicating the key role of surface cheistry in radiosensitization of AuNPs.More detailed studies suggested that such difference was inde-pendent of cellular uptake and its eficiency,but might be related to the ligand-induced difference in photoelectron generation and/or inter actions between AuNPs and X-ray triggered reactive oxygen species(ROS).

Enhanced tumor-targeted delivery of anticancer drugs by folic acid-conjugated pH-sensitive polymeric micelles

In order to enhance the targeted delivery of anticancer drugs by polymeric micelles, folic acid(FA), the ligand of folate receptor(FR) over-expressed in the most cancer cells, modified p H-sensitive polymeric micelles were designed and fabricated to encapsulate doxorubicin(DOX) by combination of p H-sensitive amphiphilic polymer poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) with FA-conjugated poly(2-ethyl-2-oxazoline)-poly(D,L-lactide). The prepared micelles were characterized to have about 36 nm in diameter with narrow distribution, well-defined spherical shape observed under TEM and p H-responsive drug release behavior. Moreover, the tumor targeting ability of the FA-modified p H-sensitive polymeric micelles was demonstrated by the cellular uptake, in vitro cytotoxicity to FR-positive KB cells and in vivo real time near-infrared fluorescence imaging in KB tumor-bearing nude mice. The efficient drug delivery by the micelles was ascribed to the synergistic effects of FR-mediated targeting and p H-triggered drug release. In conclusion, the designed FR-targeted p H-sensitive polymeric micelles might be of great potential in tumor targeted delivery of water-insoluble anticancer drugs.

Poly(L-glutamic acid)-cisplatin nanoformulations with detachable PEGylation for prolonged circulation half-life and enhanced cell internalization

PEGylation has been widely applied to prolong the circulation times of nanomedicines via the steric shielding effect,which consequently improves the intratumoral accumulation.However,cell uptake of PEGylated nanoformulations is always blocked by the steric repulsion of PEG,which limits their therapeutic effect.To this end,we designed and prepared two kinds of poly(L-glutamic acid)-cisplatin(PLG-CDDP)nanoformulations with detachable PEG,which is responsive to specific tumor tissue microenvironments for prolonged circulation time and enhanced cell internalization.The extracellular pH(pHe)-responsive cleavage 2-propionic-3-methylmaleic anhydride(CDM)-derived amide bond and matrix metalloproteinases-2/9(MMP-2/9)-sensitive degradable peptide PLGLAG were utilized to link PLG and PEG,yielding pHe-responsive PEG-pHe-PLG and MMP-sensitive PEG-MMP-PLG.The corresponding smart nanoformulations PEG-pHe-PLG-Pt and PEG-MMP-PLG-Pt were then prepared by the complexation of polypeptides and cisplatin(CDDP).The circulation half-lives of PEG-pHe-PLG-Pt and PEG-MMP-PLG-Pt were about 4.6 and 4.2 times higher than that of the control PLG-Pt,respectively.Upon reaching tumor tissue,PEG on the surface of nanomedicines was detached as triggered by pHe or MMP,which increased intratumoral CDDP retention,enhanced cell uptake,and improved antitumor efficacy toward a fatal high-grade serous ovarian cancer(HGSOC)mouse model,indicating the promising prospects for clinical application of detachable PEGylated nanoformulations.

Reducing systemic absorption and macrophages clearance of genistein by lipid-coated nanocrystals for pulmonary delivery

Pulmonary delivery is an effective drug delivery strategy for the treatment of local respiratory diseases.However,the rapid systemic absorption through the lung due to the thin barrier and persistent lung clearances influence the drug retention in the lung.In this study,we designed a lipid-coated genistein nanocrystals(Lipo-NCs)formulation to achieve enhanced efficiency of local pulmonary delivery.The LipoNCs were fabricated by modifying genistein nanocrystals(NCs)with phospholipid membrane through thin film hydration following the homogenization method.The prepared Lipo-NCs exhibited a decreased drug release rate compared with the naked NCs.Our results demonstrated that intracellular uptake and transcellular transport of NCs by the Calu-3 epithelial layer were reduced after lipid coating.Furthermore,the macrophages clearance was also impeded by this Lipo-NCs formulation.In vivo lung retention and distribution revealed that more genistein was retained in the lung after intratracheal administration of Lipo-NCs.The pharmacokinetic study displayed that the AUC((0-t))values of Lipo-NCs were 1.59-fold lesser than those of the NCs group,indicating a reduced systemic absorption.In conclusion,this research indicated that Lipo-NCs could be a suitable formulation for reducing systemic absorption and macrophages clearance,and thus enhancing drug concentration in lung by pulmonary delivery.

Mitochondria-targeted cancer therapy based on functional peptides

Mitochondria are critical for tumor growth and metastasis.A number of traditional antitumor drugs have poor water solubility and must penetrate multiple cellular barriers to reach the mitochondria.Because mitochondria have a unique transmembrane potential and an inner membrane with a low permeability,it is difficult for most drugs to enter mitochondria.In recent years,mitochondria-targeted delivery systems that use functional peptides to modify drugs have received increasing attention.Introducing functional peptides can change the original physicochemical properties of drugs and actively target mitochondria.Functional peptide-drug conjugates(PDCs,peptide-drug conjugates)can decompose and release drugs over time or due to certain stimuli in tumors.This preserves the biological activity of the drug while increasing intratumor uptake through the enhanced permeability and retention effect(EPR,the enhanced permeability and retention effect).In this review,we focus on the direction of cancer therapy and review the application of different functional peptides in the mitochondria-targeted tumor treatments reported in recent years.

Impact of particle size and pH on protein corona formation of solid lipid nanoparticles:A proof-of-concept study

When nanoparticles were introduced into the biological media,the protein corona would be formed,which endowed the nanoparticles with new bio-identities.Thus,controlling protein corona formation is critical to in vivo therapeutic effect.Controlling the particle size is the most feasible method during design,and the infuence of media pH which varies with disease condition is quite important.The impact of particle size and pH on bovine serum albumin(BSA)corona formation of solid lipid nanoparticles(SLNs)was studied here.The BSA corona formation of SLNs with increasing particle size(120-480 nm)in pH 6.0 and 7.4 was investigated.Multiple techniques were employed for visualization study,conformational structure study and mechanism study,etc."BSA corona-caused aggregation"of SLN2-3 was revealed in pH 6.0 while the dispersed state of SLNs was maintained in pH 7.4,which signifcantly affected the secondary structure of BSA and cell uptake of SLNs.The main interaction was driven by van der Waals force plus hydrogen bonding in p H 7.4,while by electrostatic attraction in pH 6.0,and size-dependent adsorption was confrmed.This study provides a systematic insight to the understanding of protein corona formation of SLNs.

Fe3O4 encapsulated mesoporous silica nanospheres with tunable size and large void pore

Magnetic Fe3O4 and mesoporous silica core- shell nanospheres with tunable size from 110-800 nm were synthesized via a one step self-assembly method. The morphological, structural, textural, and magnetic proper- ties were well-characterized by scanning electron micro- scopy, transmission electron microscopy, X-ray diffraction, N2 adsorption-desorption and magnetometer. These nanocomposites, which possess high surface area, large pore volume and well-defined pore size, exhibit two dimensional hexagonal （P6mm） mesostructures. Interest- ingly, magnetic core and mesoporous silica shell nano- composites with large void pore （20 nm） on the shell were generated by increasing the ratio of ethanol/water. Additionally, the obtained nanocomposites combined magnetization response and large void pore, implying the possibility of applications in drug/gene targeting delivery. The cell internalization capacity of NH2-functionalized nanocomposites in the case of cancer cells （HeLa cells） was exemplified to demonstrate their nano-medicine application.

Layer by layer surface engineering of poly (lactide-co-glycolide) nanoparticles:A versatile tool for nanoparticle engineering for targeted drug delivery

Recent work regarding the Layer by Layer （LbL） engineering of poly（lactide-co-glycolide） nanoparticles （PLGA NPs） is reviewed here. The LbL engineering of PLGA NPs is applied as a means of generating advanced drug delivery devices with tailored recognition, protection, cargo and release properties. LbL in combination with covalent chemistry is used to attach PEG and folic acid to control cell uptake and direct it towards cancer cells. LbL coatings composed of chitosan and alginate show low protein interactions and can be used as an alternative to Pegylation. The assembly on top of LbL coatings of lipid layers composed of variable percentages of 1,2-dioleoyl-sn-glycero-3-choline （DOPC） and 1,2-dioleoyl-sn-glycero-3-phospho- L-serine （DOPS） increases NP uptake and directs the NPs towards the endoplasmic reticulum. The antibody anti-TNF-ct is encapsulated forming a complex with alginate that is assembled LbL on top of PLGA NPs. The antibody is released in cell culture following first order kinetics. The release kinetics of encapsulated molecules inside PLGA NPs are studied when the PLGA NPs are coated via LbL with different polyelectrolytes. The intracellular release of encapsulated Doxorubicin is studied in the HepG2 cell line by means of Fluorescence Lifetime Imaging.