Cy5 fluorophore conjugation was carried as described above prior to F3 conjugation

The F3 peptide is a 31-amino acid fragment of the high mobility group protein HMG2N. F3 preferentially binds to nucleolin, a shuttle protein that is highly expressed on the plasma membranes of tumor cells.Nucleolin- targeted delivery of drug-loaded nanoparticles using F3 peptide or nucleolin-specific aptamers has been previously demonstrated.For example, F3-functionalized PEG-PLA nanoparticles led to deeper tumor penetration into 3D glioma spheroids and prolonged the survival of mouse bearing intracranial C6 glioma when loaded with paclitaxel.In this work, we conjugated F3 to TMVlys following an established protocol .In brief, TMVlys was mixed with a maleimidePEG4-NHS bifunctional linker using 10 equivalents linker per TMVlys coat protein in 10 mM KP buffer for 2 hrs. The mixture was then purified by ultra centrifugation at 112,000 g for 1 h on a 40% sucrose cushion. The F3 peptide synthesized with a C-terminal Cys was then added to the mixture at 0.5 equivalents peptide per TMVlys coat protein and reacted for 2 hrs. The final TMVlys-F3 was purified with 10,000 MW cut-off Slide-A-Lyzer MINI dialysis units. A higher excess of the F3 peptides led to aggregation and a lower excess did not show sufficient conjugation yields.

Cy5-labelled TMVlys-F3 was also prepared for cell uptake studies. Using the aforementioned protocol, SDS-NuPAGE gel electrophoresis confirmed covalent attachment of F3 peptides,bato bucket as indicated by higher molecular-weight bands The TMV coat protein has a molecular weight of ~17.5 kDa; a slightly higher molecular weight indicates the addition of F3 peptide . ImageJ software was used to quantify the degree of F3 conjugation, and data indicate that over 20% of the TMVlys coat proteins was modified with F3 peptide. The A260:A280 ratios of Cy5-TMVlys-F3 and Zn-Por:TMVlys-F3 were 1.21 and 1.29, respectively, which are indicative of intact TMV preparations . Furthermore, transmission electron microscopy indicated that TMVlys maintained its structural integrity post chemical modifications .To assess the targeting efficacy, we compared the cellular uptake of Cy5-labeled TMVlys and TMVlys-F3 particles using HeLa cells. HeLa cells were chosen because this cervical cancer cell line is known to express high levels of nucleolin.For cell targeting studies, HeLa cells were incubated with 150,000, 300,000, or 750,000 particles per cell at 37°C and 5% CO2 for 3 h in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Flow cytometry was performed as described above.

Flow cytometry showed an over 50% increase in cell uptake of conjugated Cy5-TMVlys-F3 particles . Compared to cells incubated with native TMV particles, the mean fluorescence intensity increased by 40-fold in the presence of nucleolin-targeted Cy5-TMVlys-F3 particles . Because flow cytometry does not provide insights into the fates of the nanoparticles, we also used confocal microscopy to study where the particles localized within the cells. HeLa cells were incubated with 6,000,000 particles per cell for 6 h in culture medium. Then cell membranes were stained with Alexa Fluor 555- conjugated wheat germ agglutinin and mounted with Fluoroshield™ with DAPI. Slides were imaged using a Leica TCS SPE confocal laser scanning microscope and the data was analyzed using ImageJ software. The confocal images were in agreement with flow cytometry, showing high cell interactions of the F3-targeted TMV formulation. The Cy5-TMVlys-F3 partiles appear bundled up at the surface of HeLa cells, where nucleolin is over expressed . Although others have shown intracellular trafficking of F3-functionalized polymeric nanoparticles,cellular uptake of Cy5-TMVlys-F3 was not observed in our study. This possibly indicates that the TMV’s high aspect ratio shape may not be suitable to be shuttled by nucleolin. Nevertheless, the accumulation of Cy5-TMVlys-F3 particles on the cell membrane may be advantageous for PDT as cell membrane targeting may prevent trapping of zinc porphyrin in endolysosomes and therefore enhance its cytotoxic efficacy. We moved on to explore the therapeutic efficacy of the F3-targeted Zn-Por drug delivery system. The enhanced cytotoxic efficacy of Zn-Por3+ loaded in TMVlys-F3 was reflected in the decreased survival of HeLa cells after white light treatment . TMVlys particles were loaded with 2,000:1 molar excess of Zn-Por3+ as described above prior to the conjugation of F3 peptides. From the MTT cell proliferation assay, the IC50 values for Zn-Por3+ in HeLa cells were equal to 0.034, 0.38 and 0.19 μM for Zn-Por3+ -TMVlys-F3, Zn-Por3+ -TMVlys and free Zn-Por3+ respectively. No cell killing was observed with drug-free TMVlys-F3 particles at maximum concentration.

Without light treatment, data indicated that Zn-Por3+ -TMVlys-F3 is non-toxic to HeLa cells . Compared to the data reported above, the efficacy of free Zn-Por3+ drug with white light treatment was 6-fold higher in B16F10 cells than in HeLa cells. This may be attributed to the biochemical differences between a mouse cell line and a human cell line. The drug activity decreased by half after loading into TMVlys-F3. This level of activity decrease after loading was not as significant as our previous data with TMV and TMGMV particles in B16F10 cells, yet the trend of decreased activity after loading into VNP did agree. Meanwhile, the targeted Zn-Por3+ -TMVlys-F3 particles showed a 5-fold increase in cell killing efficacy compared to the free drug. The increase PDT activity of Zn-Por3+ -TMVlys-F3 vs. Zn-Por3+ -TMVlys may be explained as follows: i) a significantly larger amount of particles targets cancer cells when using TMVlys-F3 vs. its native counterpart; and ii) TMVlys-F3 targets the cell membrane, light activation may lead to cell toxicity through cell membrane disruption, and iii) it is also possible that the PS cargo is released at the cell surface, and since the Zn-Por molecule is cell permeable and positive charged, cell uptake maybe favored – in contrast TMV without the F3 ligand accumulates in the endolysosomal compartment. In recent years, plant virus-based nanoparticles have been investigated for vaccine and immunotherapy applications to combat infectious diseases, cancers, and autoimmune diseases.Plant viruses are noninfectious to mammals and therefore are safter than their mammalian counterparts that are often used in oncolytic therapies.They can be manufactured in a cost effective manner and in large scales as viral nanoparticles as well as non-replicative viruslike particles devoid of their genomic payload.Cowpea mosaic virus VNPs and VLPs thereof have been proposed as in situ vaccine for cancer immunotherapy. The native form of CPMV consists of a bipartite ssRNA virus forming a 31 nm icosahedron with pseudo T = 3 symmetry. CPMV is made of 60 identical copies of a large and small coat proteins encapsidating RNA-1 and RNA-2 in separate but identical CPMV particles. When applied as in situ vaccine, the CPMV or eCPMV formulation is administered directly into a tumor to reprogram the tumor micro-environment and launch systemic anti-tumor immunity. While both CPMV and eCPMV demonstrated potent antitumor response in mouse models and canine patients,the RNA containing CPMV formulations demonstrated higher efficacy than eCPMV through the activation of additional cytokines and immune cells, which ultimately led to an extended survival rate of tumor-bearing mice.The proteinaceous nanoparticle presents danger signals that activate the immune system through pattern recognition receptors ,dutch bucket hydroponic and the presence of the RNA provides an additional danger signal.RNA activates TLR7/8, and induces type I IFN secretion, which increases the potency of the CPMV- based vaccines.This phenomenon was also reported using papaya mosaic virus as an in situ vaccine.While CPMV is not infectious to mammals, it remains infectious to legumes including Vigna unguiculataplants. From a translational point of view, it is thus important to develop RNA laden but non-infectious CPMV that is safe from to the environment and plants.

CPMV is stable in a variety of environmental conditions, such as temperature , pH , and in the presence of harsh chemicals, such as dimethyl sulfoxide.CPMV particles are not sensitive to certain standard methods of virus inactivation, including peptidase or hypochlorite treatment,but showed good response to ultra-violet light.Here we investigated UV treatment vs. chemical treatment of CPMV to render it non-infectious while maintaining its potent efficacy as a cancer immunotherapy. We compared β-propiolactone or formalin treatment with the previously reported UV inactivation method. These chemical treatment modalities are commonly used to produce non-virulent vaccines such as polio, hepatitis A, enterovirus, and influenza viruses vaccines.Of particular interests, these methods do not remove the RNA from the VNP, but rather create RNA damage preventing its transcription and translation, and therefore viral replication. UV light promotes RNA-protein crosslinking and dimerization of adjacent urcacils.βPL promotes the alkylation or acylation of cytidine, deoxyadenosine, and deoxyguanosine. βPL treatment also leads to a large extend of protein modifications; for example poliovirus proteins are more extensively modified by βPL than nucleic acid during viral inactivation.Specifically, βPL acylates and alkylates to a great extent cysteine, histidine, and methionine, and to a lesser extent with aspartic acid, glutamic acid, lysine, serine, threonine, and tyrosine.Lastly, formalin causes protein-protein and RNA-protein covalent crosslinking.To gain further insights into whether and to what degree RNA-protein crosslinking occurred, intact and denatured CPMV particles pre- and post-inactivation were analyzed by native gel electrophoresis and denatured samples were analyzed by SDS-PAGE. All gels were imaged after staining for proteins and nucleic acid under white light and UV light, respectively. In the native agarose GE, two distinctive protein bands were observed due to the presence of two electrophoretic forms of CPMV particles ; the fast form of CPMV is the result of a 24 amino acid loss at the C-terminus of the small coat protein due to proteolysis in plant cells.In all samples, nucleic acids and proteins traveled through the agarose gel together, confirming the entrapment of the RNA within the CPMV particles. CPMV treated with doses of 5, 7.5, 10 and 15 J cm-2 of UV showed signs of particle aggregation, as indicated by the presence of a smear instead of two distinct bands. Compared to CPMV, βPL-CPMV and Form-CPMV traveled farther through the gel proportionally to the concentration of βPL and formalin employed to inactivate CPMV. These results could reflect a change in overall particle charge due to the action of βPL and formalin on proteins and RNA. Samples were denatured by SDS-PAGE to separate the L and S coat protein subunits, visualized as single bands at 42 kDa and 24 kDa, respectively . The band intensity of UV-CPMV coat proteins decreased with increasing dose of UVradiation, indicative of the presence of particle aggregates. βPL-CPMV showed no sign of protein breakage or aggregation regardless of the dose of βPL used during treatment. In contrast, the higher the dose of formalin, the more inter-CP cross linking was observed at doses as low as 0.5 mM . GelRed staining was added to SDS-PAGE gels to assess the integrity of the RNA content of particles. RNA from UV-CPMV and Form-CPMV particles did not travel through the gel, most likely due to intra-RNA and RNA-coat protein cross linking; the samples remained stuck in the wells. RNA from βPL-CPMV matched the profile of that released from CPMV; however, at high βPL concentrations RNA breakage was observed – this is also consistent with UV/visible spectroscopy and SEC data as described above. Therefore, data indicate that at doses higher than 1 J cm-2 UV light or 1 mM βPL or formalin RNA and protein modifications and crosslinking occur; at high concentration of UV light, CPMV particle aggregation is observed; and at high βPL concentrations, RNA breakage is indicated. Agarose gel electrophoresis confirmed that leaves infected with CPMV inactivated through UV treatment at doses of 7.5 J cm- 2 or higher were indeed effectively inactivated and CPMV infection was not detectable by RTPCR . Similarly, formulations treated with 50 mM βPL-CPMV and 1 mM FormCPMV were confirmed to be fully inactivated. It is worth mentioning that leaves inoculated with 10 mM βPL-CPMV showed no visual symptoms of infection, yet these leaves tested positive by RT-PCR – the latter is a more sensitive assay. Based on the inactivation studies and infection assays in plants, for all subsequent experiments, we used the inactivated CPMV formulation obtained from 7.5 J cm-2 UV, 50 mM βPL, and 1 mM formalin. At these doses UV-CPMV was structurally intact but to some degree aggregated, and there was evidence of RNA-RNA and RNA-protein crosslinking. βPL-CPMV remained structurally sound and monodisperse but its RNA was severely damaged. FormCPMV also retained its size and monodispersity even though protein-protein, protein-RNA, and RNA-RNA cross linking occurred.