Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides

Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy.     

Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery

The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L.-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supereoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA. while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlledrelease delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.     

pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles. Poly(L-histidine) ?PLH? has been shown to induce membrane fusion at endosomal pH values, whereas PLL has a well documented efficacy in polyplex formation. Therefore, N-Ac-poly(L-histidine)-graft-poly(L-lysine) ?PLH-g-PLL? was synthesized by grafting poly(L-histidine) to poly(L-lysine) ?PLL?. PLH-g-PLL formed polyplex particles by electrostatic interactions with plasmid DNA ?pDNA?. The mean particle size of the polyplexes was in the range of 117 +/- 6 nm to 306 +/- 77 nm. PLH-g-PLL gene carrier demonstrated higher transfection efficacy in 293T cells than PLL at all equivalent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic agent enhanced transfection for both PLL and PLH-g-PLL gene carriers. PLH-g-PLL enhanced beta-galactosidase expression compared to PLL, but still increased in efficacy when chloroquine was included.     

The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression

BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.     

Glycopolymer modification on physicochemical and biological properties of poly(L-lysine) for gene delivery

Poly(L-lysine) (PLL) has excellent plasmid DNA (pDNA) condensation capacity. However, the relatively high cytotoxicity and low transfection efficiency limit its application as gene delivery vectors. Here, well-defined glycopolymers are synthesized by reversible addition fragmentation transfer polymerization and grafted onto PLL to improve the gene delivery performance. After glycopolymer modification, PLL shows reduced cytotoxicity. By regulating the glycopolymer length and amino group substitution degree, the glycopolymer modified PLL can condense pDNA with proper strength, protect the condensed pDNA from degradation and release them in time. Transfection with NIH3T3 and HepG2 cells shows that the glycopolymer modified PLL has improved transfection efficiencies. The low cytotoxicity, effective pDNA protection and enhanced transfection efficiencies indicate that glycopolymer modification would be an effective strategy to improve the polycation properties for gene delivery.     

Gene delivery using biodegradable polyelectrolyte microcapsules prepared through the layer-by-layer technique

Biodegradable and non‐biodegradable microcapsules were prepared via the layer‐by‐layer (LbL) technique consisting of the polyelectrolyte pairs of dextran sulphate/poly‐L ‐arginine and poly(styrene sulfonate)/poly(allylamine hydrochloride), respectively, in an attempt to encapsulate plasmid DNA (pDNA) for efficient transfection into NIH 3T3 cells. Results indicated the retention of bioactivity in the encased pDNA, as well as a correlation between the level of in vitro gene expression and biodegradability properties of polyelectrolyte. Furthermore, the incorporation of iron oxide nanoparticles within the polyelectrolyte layers significantly improved the in vitro transfection efficiency of the microcapsules. As a novel pDNA delivery system, the reported biodegradable microcapsules provide useful insight into plasmid‐based vaccination and where there is a prerequisite to deliver genes into cells capable of phagocytosis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1088–1094, 2012     

Thermal hysteresis proteins

Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.     

Folic acid conjugated amino acid-based star polymers for active targeting of cancer cells

Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.     

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25 kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.     

Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.     

Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions

The stability in physiological medium of polyplex- and lipoplex-type nonviral gene vectors was evaluated by detecting the conformational change of complexed plasmid DNA (pDNA) labeled simultaneously with fluorescein (energy donor) and X-rhodamine (energy acceptor) through fluorescence resonance energy transfer (FRET). Upon mixing with cationic components, such as LipofectAMINE, poly(L-lysine), and poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys), the fluorescence spectrum of doubly labeled pDNA underwent a drastic change due to the occurrence of FRET between the donor-acceptor pair on pDNA taking a globular conformation (condensed state) through complexation. The measurement was carried out also in the presence of 20% serum, under which conditions FRET from condensed pDNA was clearly monitored without interference from coexisting components in the medium, allowing evaluation of the condensed state of pDNA in nonviral gene vectors under physiological conditions. Serum addition immediately induced a sharp decrease in FRET for the LipofectAMINE/pDNA (lipoplex) system, which was consistent with the sharp decrease in the transfection efficiency of the lipoplex system in serum-containing medium. In contrast, the PEG-PLys/pDNA polyplex (polyion complex micelle) system maintained appreciable transfection efficiency even in serum-containing medium, and FRET efficiency remained constant for up to 12 h, indicating the high stability of the polyion complex micelle under physiological conditions.     

Intracellular delivery of nucleic acid by cell-permeable hPP10 peptide

Although gene therapy offers hope against incurable diseases, nonreplicating transduction vectors remain lacking. We have previously characterized a cell-penetrating peptide hPP10 for the delivery of various cargoes; however, whether hPP10 can mediate nucleic acid delivery is still unknown. Here, examining via different ways, we demonstrate that hPP10 stably complexes with plasmid DNA (pDNA) and safely mediates nucleic acid transfection. hPP10 can mediate GFP-, dsRed-, and luciferase-expressing plasmids into cells with nearly the same efficiency as commercial transfection reagents Turbofectin or Lipofect. Furthermore, hPP10 can mediate Cre fusion protein delivery and pDNA transfection simultaneously in the Cre/loxp system in vitro. In addition, hPP10 fused with an RNA-binding domain can mediate delivery of small interfering RNA into cells to silence the reporter gene expression. Collectively, our results suggest that hPP10 is an option for nucleic acid delivery with efficiencies similar to that of commercial reagents.     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

Amphiphilic block copolymers enhance cellular uptake and nuclear entry of polyplex-delivered DNA

This work for the first time demonstrates that synthetic polymers enhance uptake and nuclear import of plasmid DNA (pDNA) through the activation of cellular trafficking machinery. Nonionic block copolymers of poly(ethylene oxide) and poly(propylene oxide), Pluronics, are widely used as excipients in pharmaceutics. We previously demonstrated that Pluronics increase the phosphorylation of IkappaB and subsequent NFkappaB nuclear localization as well as upregulate numerous NFkappaB-related genes. In this study, we show that Pluronics enhance gene transfer by pDNA/polycation complexes ("polyplexes") in a promoter-dependent fashion. Addition of Pluronic P123 or P85 to polyethyleneimine-based polyplexes had little effect on polyplex particle size but significantly enhanced pDNA cellular uptake, nuclear translocation, and gene expression in several cell lines. When added to polyplex-transfected cells after transfection, Pluronics enhanced nuclear import of pDNA containing NFkappaB binding sites, but have no effect on import of pDNA without these sites. Altogether, our studies suggest that Pluronics rapidly activate NFkappaB, which binds cytosolic pDNA that possesses promoters containing NFkappaB binding sites and consequently increase nuclear import of pDNA through NFkappaB nuclear translocation.     

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N′-cystamine-bis-acrylamide)/pDNA complexes

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)—25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N′-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.     

Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation

Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.     

Gene Transfer by DNA/mannosylated Chitosan Complexes into Mouse Peritoneal Macrophages

Chitosan is a biodegradable and biocompatible polymer and is useful as a non-viral vector for gene delivery. In order to deliver pDNA/chitosan complex into macrophages expressing a mannose receptor, mannose-modified chitosan (man-chitosan) was employed. The cellular uptake of pDNA/man-chitosan complexes through mannose recognition was then observed. The pDNA/man-chitosan complexes showed no significant cytotoxicity in mouse peritoneal macrophages, while pDNA/man-PEI complexes showed strong cytotoxicity. The pDNA/man-chitosan complexes showed much higher transfection efficiency than pDNA/chitosan complexes in mouse peritoneal macrophages. Observation with a confocal laser microscope suggested differences in the cellular uptake mechanism between pDNA/chitosan complexes and pDNA/man-chitosan complexes. Mannose receptor-mediated gene transfer thus enhances the transfection efficiency of pDNA/chitosan complexes.     

Nuclear delivery of plasmid DNA determines the efficiency of gene expression

As a cationic non‐viral gene delivery vector, poly(agmatine/ N, N′‐cystamine‐bis‐acrylamide) (AGM‐CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM‐CBA/pDNA polyplexes was found to have a non‐linear relationship with AGM‐CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM‐CBA), we used pGL3‐control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM‐CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate‐limiting step for pLUC transfection expression. Further optimization of the non‐viral gene delivery system can be focused on the improvement of gene intracellular availability.     

Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.