Needleless administration of advanced therapies into the skin via the appendages using a hypobaric patch.

Advanced therapies are commonly administered via injection even when they act within the skin tissue, and this increases the chances of off-target effects. Here we report the use of a skin patch containing a hypobaric chamber that induces skin dome formation to enable needleless delivery of advanced therapies directly into porcine, rat, and mouse skin. Finite element method modeling showed that the hypobaric chamber in the patch opened the skin appendages by 32%, thinned the skin, and compressed the appendage wall epithelia. These changes allowed direct delivery of an H1N1 vaccine antigen and a diclofenac nanotherapeutic into the skin. Fluorescence imaging and infrared mapping of the skin showed needleless delivery via the appendages. The in vivo utility of the patch was demonstrated by a superior immunoglobulin G response to the vaccine antigen in mice compared to intramuscular injection and a 70% reduction in rat paw swelling in vivo over 5 h with diclofenac without skin histology changes.

PET Imaging of Small Extracellular Vesicles via [89Zr]Zr(oxinate)4 Direct Radiolabeling.

Exosomes or small extracellular vesicles (sEVs) are increasingly gaining attention for their potential as drug delivery systems and biomarkers of disease. Therefore, it is important to understand their in vivo biodistribution using imaging techniques that allow tracking over time and at the whole-body level. Positron emission tomography (PET) allows short- and long-term whole-body tracking of radiolabeled compounds in both animals and humans and with excellent quantification properties compared to other nuclear imaging techniques. In this report, we explored the use of [89Zr]Zr(oxinate)4 (a cell and liposome radiotracer) for direct and intraluminal radiolabeling of several types of sEVs, achieving high radiolabeling yields. The radiosynthesis and radiolabeling protocols were optimized for sEV labeling, avoiding sEV damage, as demonstrated using several characterizations (cryoEM, nanoparticle tracking analysis, dot blot, and flow cytometry) and in vitro techniques. Using pancreatic cancer sEVs (PANC1) in a healthy mouse model, we showed that it is possible to track 89Zr-labeled sEVs in vivo using PET imaging for at least up to 24 h. We also report differential biodistribution of intact sEVs compared to intentionally heat-damaged sEVs, with significantly reduced spleen uptake for the latter. Therefore, we conclude that 89Zr-labeled sEVs using this method can reliably be used for in vivo PET tracking and thus allow efficient exploration of their potential as drug delivery systems.

Defined serum-free three-dimensional culture of umbilical cord-derived mesenchymal stem cells yields exosomes that promote fibroblast proliferation and migration in vitro.

Stem cell-derived exosomes are emerging as novel and clinically relevant cell-free therapeutics for regenerative therapy. This work focused on investigating the stimulation of fibroblasts by exosomes derived from umbilical cord-derived mesenchymal stem cells (ucMSC) in a defined serum-free three-dimensional (3D) culture. 3D culture of ucMSC was carried out in medium supplemented with KnockOut serum replacement (KO-medium) using the Aggrewell system. ucMSC in KO-medium formed spheroids with maintained size and integrity throughout culture. This enabled the isolation of vesicles from ucMSC spheroids in KO-medium with sizes that fall within the exosomal size range and were positive for the expression of canonical exosomal markers CD63, CD9, CD81, Alix, and TSG101. The ucMSC-derived exosomes (ExoucMSC ) were shown to significantly increase the migration and proliferation of murine fibroblasts in vitro. To conclude, 3D culture of ucMSC in defined serum-free KO-medium formed viable spheroids which enabled the isolation of ExoucMSC with the potential of accelerating wound healing.

Magnetic Drug Targeting: Preclinical in Vivo Studies, Mathematical Modeling, and Extrapolation to Humans.

A sound theoretical rationale for the design of a magnetic nanocarrier capable of magnetic capture in vivo after intravenous administration could help elucidate the parameters necessary for in vivo magnetic tumor targeting. In this work, we utilized our long-circulating polymeric magnetic nanocarriers, encapsulating increasing amounts of superparamagnetic iron oxide nanoparticles (SPIONs) in a biocompatible oil carrier, to study the effects of SPION loading and of applied magnetic field strength on magnetic tumor targeting in CT26 tumor-bearing mice. Under controlled conditions, the in vivo magnetic targeting was quantified and found to be directly proportional to SPION loading and magnetic field strength. Highest SPION loading, however, resulted in a reduced blood circulation time and a plateauing of the magnetic targeting. Mathematical modeling was undertaken to compute the in vivo magnetic, viscoelastic, convective, and diffusive forces acting on the nanocapsules (NCs) in accordance with the Nacev-Shapiro construct, and this was then used to extrapolate to the expected behavior in humans. The model predicted that in the latter case, the NCs and magnetic forces applied here would have been sufficient to achieve successful targeting in humans. Lastly, an in vivo murine tumor growth delay study was performed using docetaxel (DTX)-encapsulated NCs. Magnetic targeting was found to offer enhanced therapeutic efficacy and improve mice survival compared to passive targeting at drug doses of ca. 5-8 mg of DTX/kg. This is, to our knowledge, the first study that truly bridges the gap between preclinical experiments and clinical translation in the field of magnetic drug targeting.

The Shortening of MWNT-SPION Hybrids by Steam Treatment Improves Their Magnetic Resonance Imaging Properties In Vitro and In Vivo.

Carbon nanotubes (CNTs) have been advocated as promising nanocarriers in the biomedical field. Their high surface area and needle-like shape make these systems especially attractive for diagnostic and therapeutic applications. Biocompatibility, cell internalization, biodistribution, and pharmacokinetic profile have all been reported to be length dependent. In this study, further insights are gotten on the role that the length of CNTs plays when developing novel contrast agents for magnetic resonance imaging (MRI). Two samples of CNTs with different length distribution have been decorated with radio-labeled iron oxide nanoparticles. Despite characterization of the prepared hybrids reveals a similar degree of loading and size of the nanoparticles for both samples, the use of short CNTs is found to enhance the MRI properties of the developed contrast agents both in vitro and in vivo compared to their long counterparts.

Passively Targeted Curcumin-Loaded PEGylated PLGA Nanocapsules for Colon Cancer Therapy In Vivo.

Clinical applications of curcumin for the treatment of cancer and other chronic diseases have been mainly hindered by its short biological half-life and poor water solubility. Nanotechnology-based drug delivery systems have the potential to enhance the efficacy of poorly soluble drugs for systemic delivery. This study proposes the use of poly(lactic-co-glycolic acid) (PLGA)-based polymeric oil-cored nanocapsules (NCs) for curcumin loading and delivery to colon cancer in mice after systemic injection. Formulations of different oil compositions are prepared and characterized for their curcumin loading, physico-chemical properties, and shelf-life stability. The results indicate that castor oil-cored PLGA-based NC achieves high drug loading efficiency (≈18% w(drug)/w(polymer)%) compared to previously reported NCs. Curcumin-loaded NCs internalize more efficiently in CT26 cells than the free drug, and exert therapeutic activity in vitro, leading to apoptosis and blocking the cell cycle. In addition, the formulated NC exhibits an extended blood circulation profile compared to the non-PEGylated NC, and accumulates in the subcutaneous CT26-tumors in mice, after systemic administration. The results are confirmed by optical and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. In vivo growth delay studies are performed, and significantly smaller tumor volumes are achieved compared to empty NC injected animals. This study shows the great potential of the formulated NC for treating colon cancer.

Design of Cationic Multiwalled Carbon Nanotubes as Efficient siRNA Vectors for Lung Cancer Xenograft Eradication.

Polo-Like Kinase (PLK1) has been identified as a potential target in cancer gene therapy via chemical or genetic inhibitory approaches. The biomedical applications of chemically functionalized carbon nanotubes (f-CNTs) in cancer therapy have been studied due to their ability to efficiently deliver siRNA intracellularly. In this study, we established the capacity of cationic MWNT-NH3(+) to deliver the apoptotic siRNA against PLK1 (siPLK1) in Calu6 tumor xenografts by direct intratumoral injections. A direct comparison with cationic liposomes was made. This study validates the PLK1 gene as a potential target in cancer gene therapy including lung cancer, as demonstrated by the therapeutic efficacy of siPLK1:MWNT-NH3(+) complexes and their ability to significantly improve animal survival. Biological analysis of the siPLK1:MWNT-NH3(+) treated tumors by qRT-PCR and Western blot, in addition to TUNEL staining confirmed the biological functionality of the siRNA intratumorally, suggesting that tumor eradication was due to PLK1 knockdown. Furthermore, by using a fluorescently labeled, noncoding siRNA sequence complexed with MWNT-NH3(+), we established for the first time that the improved therapeutic efficacy observed in f-CNT-based siRNA delivery is directly proportional to the enhanced siRNA retention in the solid tumor and subsequent uptake by tumor cells after local administration in vivo.

Cationic Liposome- Multi-Walled Carbon Nanotubes Hybrids for Dual siPLK1 and Doxorubicin Delivery In Vitro.

PURPOSE: To formulate f-MWNTs-cationic liposome hybrids for the simultaneous delivery of siPLK1 and doxorubicin to cancer cells. METHOD: f-MWNTs-cationic liposome hybrids were prepared by the thin film hydration method where the lipid film was hydrated with 100 µg/ml or 1 mg/ml of ox-MWNTs-NH3 (+) or MWNTs-NH3 (+) in 5% dextrose. siRNA complexation and protection ability was determined by agarose gel electrophoresis. f-MWNTs and liposome interaction was evaluated using Nile Red (NR) fluorescence spectroscopy. Cellular uptake in A549 cells was assessed by flow cytometry. Silencing of target proteins was determined by Luciferase and MTT assays. Sub-G1 analysis was performed to evaluate apoptosis following co-delivery of siPLK1 and Doxorubicin (Dox). RESULTS: Zeta potential and siRNA complexation profile obtained for all hybrids were comparable to those achieved with cationic liposomes. ox-MWNTs-NH3 (+) showed greater extent of interaction with cationic liposomes compared to MWNTs-NH3 (+). ox-MWNTs-NH3 (+) was able to protect siRNA from nuclease-mediated degradation. Enhanced cellular uptake of both the carrier and loaded siRNA in A549 cell, were observed for this hybrid compared to the liposomal carrier. A synergistic pro-apoptotic effect was obtained when siPLK1 silencing was combined with doxorubicin treatment for the hybrid:siRNA complexes compared to the lipoplexes, in A549 cells in vitro. CONCLUSIONS: f-MWNTs-cationic liposome hybrid designed in this study can serve as a potential vehicle for the co-delivery of siRNA and cytotoxic drugs to cancer cells in vitro.

Mutation of arginine residues to avoid non-specific cellular uptakes for hepatitis B virus core particles.

BACKGROUND: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues. RESULTS: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells. CONCLUSIONS: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

Magnetically Decorated Multi-Walled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents.

Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.

Production of water-soluble few-layer graphene mesosheets by dry milling with hydrophobic drug.

A novel, fast, and easy mechano-chemistry-based (dry milling) method has been developed to exfoliate graphene with hydrophobic drugs generating few-layer graphene mesosheets (< 10 nm in thickness and ∼1 µm in width). The electronic properties of the graphitic structure were partially preserved after the milling treatment compared with graphene oxide prepared by Hummers' method. Several characterization techniques such as thermogravimetric analysis, Raman spectroscopy, atomic force microscopy, electron microscopy, and molecular dynamics simulation were used to characterize this material. The drug-exfoliated mesosheets were pharmacologically inactive, offering a new approach for making water-soluble few-layer graphene mesosheets upon dry milling with hydrophobic drugs, mainly used as exfoliating agents.

Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing.

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.

Plasmid DNA ionisable lipid nanoparticles as non-inert carriers and potent immune activators for cancer immunotherapy.

Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo. In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.

Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent.

Extracellular vesicles (EVs) derived from mesenchymal stem cells are promising nanotherapeutics in liver diseases due to their regenerative and immunomodulatory properties. Nevertheless, a concern has been raised regarding the rapid clearance of exogenous EVs by phagocytic cells. Here we explore the impact of protein corona on EVs derived from two culturing conditions in which specific proteins acquired from media were simultaneously adsorbed on the EV surface. Additionally, by incubating EVs with serum, simulating protein corona formation upon systemic delivery, further resolved protein corona-EV complex patterns were investigated. Our findings reveal the potential influences of corona composition on EVs under in vitro conditions and their in vivo kinetics. Our data suggest that bound albumin creates an EV signature that can retarget EVs from hepatic macrophages. This results in markedly improved cellular uptake by hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells. This phenomenon can be applied as a camouflage strategy by precoating EVs with albumin to fabricate the albumin-enriched protein corona-EV complex, enhancing non-phagocytic uptake in the liver. This work addresses a critical challenge facing intravenously administered EVs for liver therapy by tailoring the protein corona-EV complex for liver cell targeting and immune evasion.

Ammonium and guanidinium dendron-carbon nanotubes by amidation and click chemistry and their use for siRNA delivery.

A series of multi-walled carbon nanotube (MWCNT) conjugates is described, functionalized with different dendrons bearing positive charges at their termini (i.e. ammonium or guanidinium groups). The dendrimeric units are anchored to the nanotube scaffolds using two orthogonal synthetic approaches, amidation and click reactions. The final nanohybrids are characterized by complementary analytical techniques, while their ability to interact with siRNA is investigated by means of agarose gel electrophoresis. The demonstration of the cell uptake capacity, the low cytotoxicity, and the ability of these cationic conjugates to silence cytotoxic genes suggests them to be promising carriers for genetic material.

Inhalable DNase I microparticles engineered with biologically active excipients.

Highly viscous mucus poses a big challenge for the delivery of particulates carrying therapeutics to patients with cystic fibrosis. In this study, surface modifying DNase I loaded particles using different excipients to achieve better lung deposition, higher enzyme stability or better biological activity had been exploited. For the purpose, controlled release microparticles (MP) were prepared by co-spray drying DNase I with the polymer poly-lactic-co-glycolic acid (PLGA) and the biocompatible lipid surfactant 1,2-dipalmitoyl-Sn-phosphatidyl choline (DPPC) using various hydrophilic excipients. The effect of the included modifiers on the particle morphology, size, zeta potential as well as enzyme encapsulation efficiency, biological activity and release had been evaluated. Powder aerosolisation performance and particle phagocytosis by murine macrophages were also investigated. The results showed that more than 80% of enzyme activity was recovered after MP preparation and that selected surface modifiers greatly increased the enzyme encapsulation efficiency. The particle morphology was greatly modified altering in turn the powders inhalation indices where dextran, ovalbumin and chitosan hydrochloride increased considerably the respirable fraction compared to the normal hydrophilic carriers lactose and PVP. Despite of the improved aerosolisation caused by chitosan hydrochloride, yet retardation of chitosan coated particles in artificial mucus samples discouraged its application. On the other hand, dextran and polyanions enhanced DNase I effect in reducing cystic fibrosis mucus viscosity. DPPC proved good ability to reduce particles phagocytic uptake even in the presence of the selected adjuvants. The prepared MP systems were biocompatible with lung epithelial cells. To conclude, controlled release DNase I loaded PLGA-MP with high inhalation indices and enhanced mucolytic activity on CF sputum could be obtained by surface modifying the particles with PGA or dextran.

Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth.

This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G(6)) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems.

Combined Facile Synthesis, Purification, and Surface Functionalization Approach Yields Monodispersed Gold Nanorods for Drug Delivery Applications.

Synthesizing gold nanorods (AuNRs) by seed-mediated growth method results in the presence of undesired size and shape particles by-products occupying 10-90% of the population. In this study, AuNRs are synthesized by the seed-mediated growth method using cetyltrimethylammonium bromide (CTAB) as a surfactant. AuNRs with redshifted longitudinal localized surface plasmon resonance (LLSPR) peak, localized in the biological "transparency window" (650-1350 nm), are synthesized after optimizing seed solution, silver nitrate solution, and hydrochloric acid solution volumes, based on the published protocols. A two-step purification method, dialysis followed by centrifugation, is applied to remove excess CTAB and collect LLSPR-redshifted AuNRs with high rod purity (>90%). CTAB is subsequently exchanged with polyethylene glycol (PEG) to improve AuNRs biocompatibility. PEGylated AuNRs are confirmed innocuous to both SN4741 cells and B16F10 cells by the modified MTT assay and the modified lactate dehydrogenase (LDH) assay up to 1 nm and 24 h incubation. In this study, a combined facile synthesis, purification, and surface functionalization approach is proposed to obtain water-dispersible monodispersed AuNRs for drug delivery applications.

Plasmid DNA cationic non-viral vector complexes induce cytotoxicity-associated PD-L1 expression up-regulation in cancer cells in vitro.

Non-viral vectors are promising nucleic acid carriers which have been utilized in gene-based cancer immunotherapy. The aim of this study is to compare the transfection efficiency and cytotoxicity of three cationic non-viral vectors namely Polyethylenimine (PEI), Lipofectamine 2000 (LPF) and stable nucleic acid lipid particles (SNALPs) of different lipid compositions, for the delivery of plasmid DNA (pDNA) expressing immunostimulatory molecules, OX40L or 4-1BBL, to cancer cells in vitro. The results indicate that PEI and LPF are efficient vectors for pDNA delivery with high transfection efficiency obtained. However, pDNA-PEI and pDNA-LPF complexes up-regulated the expression of programmed death ligand-1 (PD-L1) and induced significant cytotoxicity in both B16F10 and CT26 cell lines. The up-regulation of PD-L1 expression induced by pDNA-PEI and pDNA-LPF complexes was independent of cancer cell line, nor was it linked to the presence of GpC motifs in the pDNA. In contrast, the use of biocompatible SNALPs (MC3 and KC2 types) resulted in lower pDNA transfection efficiency, however no significant up-regulation of PD-L1 or cytotoxicity was observed. A strong correlation was found between up-regulation of PD-L1 expression and cytotoxicity. Up-regulation of PD-L1 expression could be mitigated with RNAi, maintaining expression at basal levels. Due to the improved biocompatibility and the absence of PD-L1 up-regulation, SNALPs represent a viable non-viral nucleic acid vector for delivery of pDNA encoding immunostimulatory molecules. The results of this study suggest that PD-L1 expression should be monitored when selecting commercial transfection reagents as pDNA vectors for cancer immunotherapy in vitro.

Anti-tumor antibody isotype response can be modified with locally administered immunoadjuvants.

In situ vaccination with immunostimulatory compounds is a demonstrated means to treat tumors preclinically. While these therapeutic effects have been attributed to the actions of T cells or innate immune activation, characterisation of the humoral immune response is seldom performed. This study aims to identify whether the injection of immunoadjuvants, Addavax (Adda) and cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG), intratumorally can influence the antibody response. Specifically, whether intratumoral injection of immunoadjuvants can alter the tumor-specific antibody target, titre and isotype. Following this, the study aimed to investigate whether serum obtained from in situ vaccinated mice could neutralise circulating tumor cells. Serum was obtained from mice bearing B16F10-OVA-Luc-GFP tumors treated with immunoadjuvants. Antibody targets' titre and isotype were assessed by indirect ELISA. The ability of serum to neutralise circulating cancer cells was evaluated in a B16F10 pseudo-metastatic model. It was observed that tumor-bearing mice mount a specific anti-tumor antibody response. Antibody titre and target were unaffected by in situ vaccination with immunoadjuvants; however, a higher amount of IgG2c was produced in mice receiving Adda plus CpG. Serum from in situ vaccinated mice was unable to neutralise circulating B16F10 cells. Thus, this study has demonstrated that anti-tumor antibody isotype may be modified using in situ vaccination; however, this alone is not sufficient to neutralise circulating cancer cells.