IntroductionThe Toll-like receptor (TLR) 7/8 agonist resiquimod has shown promise for precancerous lesions of cutaneous squamous cell carcinoma (cSCC) but remains unexplored as a treatment for cSCC. Additionally, ablative fractional laser (AFL) has been shown to enhance the efficacy of TLR7 agonist in mouse tumor models. This study investigates the efficacy of intratumoral resiquimod formulated into a sustained-release gel (RSQ-gel) in a cSCC mouse model and compares RSQ-gel with topical imiquimod (IMQ) cream, a clinically approved TLR7 agonist. We further examine whether adjuvant AFL enhances the efficacy of RSQ-gel.MethodsA syngeneic transplanted cSCC mouse model was established using cells from a UVR-induced cSCC mouse model. The immunostimulatory effects of RSQ-gel were assessed by analyzing the expression of the activation marker CD86 on plasmacytoid dendritic cells (pDC) and cross-presenting conventional type I dendritic cells (XCR1+ cDC1) via flow cytometry. Tumor growth and survival outcomes were evaluated for RSQ-gel as monotherapy and in combination with AFL.ResultsRSQ-gel was associated with activation of pDCs and XCR1+ cDC1s in the tumor-draining lymph node, as indicated by higher expression of CD86 compared to IMQ (P< 0.0001, P = 0.00175, respectively). RSQ-gel monotherapy delayed tumor growth but did not prolong survival (P = 0.0651). However, combining RSQ-gel with AFL resulted in prolonged survival compared to AFL-treated and untreated mice (P = 0.0153, P = 0.0214, respectively). Weekly RSQ-gel treatment induced comparable efficacy to daily topical IMQ treatment.DiscussionRSQ-gel with AFL demonstrated significant antitumor efficacy in the cSCC mouse model. Local RSQ-gel combined with adjuvant AFL may offer a promising therapeutic approach for cSCC.

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice.

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.

Purpose: Drug delivery to the retina remains a challenge due to ocular barriers and fast clearing mechanisms. Nanocarrier drug delivery systems (NDDSs) hold the promise of prolonging intraocular retention times and increasing drug concentrations in the retina. Methods: Anionic and cationic PEGylated liposomes, loaded with oxaliplatin (OxPt) to be used as trace element, were prepared from dry lipid powders. The differently charged liposomes were intravitreally injected in C57BL/6JrJ mice; eyes were harvested 2 hours and 24 hours post-injection. To investigate active transport mechanisms in the eye, a subset of mice were pre-injected with chloroquine before injection with cationic liposomes. Eyes were dissected and the distribution of OxPt in different tissues were quantified by inductively coupled plasma mass spectrometry (ICP-MS). Results: Both liposome formulations enhanced the retention time of OxPt in the vitreous over free OxPt. Surprisingly, when formulated in cationic liposomes, OxPt translocated through the retina and accumulated in the RPE-sclera. Pre-injection with chloroquine inhibited the transport of liposomal OxPt from the vitreous to the RPE-sclera. Conclusions: We show that liposomes can enhance the retention time of small molecular drugs in the vitreous and that active transport mechanisms are involved in the trans retinal transport of NDDS after intravitreal injections. Translational Relevance: These results highlight the need for understanding the dynamics of ocular transport mechanisms in living eyes when designing NDDS with the back of the eye as the target. Active transport of nanocarriers through the retina will limit the drug concentration in the neuronal retina but might be exploited for targeting the RPE.

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic, immunosuppressive TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming a sustained drug release depot at the injection site that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues to allow infiltration of T cells, consequently generating systemic anticancer immunoreactivity. The CarboCell technology can release multiple small molecule drugs - each with tailored release profiles - rendering it active across the broad composition of TME backgrounds. In the current study, impressive therapeutic performance is presented for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor. CarboCell can be injected through standard clinical thin-needle technologies. Its inherent magnetic resonance imaging and ultrasound visibility, and optional radiographic contrast, make it possible to validate and plan CarboCell injections across clinical imaging modalities. These features, in combination with attractive injection intervals, secure optimal patient compliance and open new possibilities for intratumoral immunotherapy accurately across basically all anatomical locations.

Abstract Intratumoral immune activating therapy is biologically supported by the cancer immune response being generated in the tumor microenvironment (TME) and draining lymph nodes. Successful intratumoral immune activation is however challenging as the cells in the TME are plastic and reactive, and therefore requires continuous stimulation. This is problematic as drugs are rapidly washed out of tumors, and frequent intratumoral injections limit manageable locations, patient compliance and provides risks for cancer cell dissemination. The CarboCell technology overcomes this by forming a positionally stable drug release depot at the injection site with tailored release profiles of single or multiple drugs. Here we demonstrate the therapeutic performance of a CarboCell formulation releasing a TLR7/8 agonist and a TGFb inhibitor (CarboCell TLR:TGFb). CarboCell provides intratumoral drug retention up to 90% at day 3 and 50% at day 7 after injection in mouse tumors and beyond 14 days in canines. By control of the release kinetics, more than five times the systemically tolerated drug dose was tolerated for intratumoral CarboCell TLR:TGFb even at repeated dosing. CarboCell TLR:TGFb combinatorial therapy provided complete tumor rejection in 71%, 67% and 89% of mice carrying large MC38, EMT-6 and CT26 tumors. The dual drug CarboCell TLR:TGFb formulation was therapeutically superior to single drug CarboCell formulations, which underlines the synergistic value the combinatorial therapy. CarboCell TLR:TGFb was further demonstrated to inhibit lung metastasis in highly metastatic cancer models. Mechanistically, CarboCell TLR:TGFb activated the innate and adaptive immune system. Inflammatory cytokines were rapidly induced and accompanied by cancer cells necroptosis. This was followed by an increased number and activation of central DCs in the tumor draining lymph node and anti-cancer specific T cells in the tumor. CarboCell TLR:TGFb demonstrate potent immune activating properties and overcomes the multifactorial immunosuppressive barriers in the TME. Clinically attractive features of CarboCell further includes intrinsic MR and US visibility and radiographic contrast. This, in combination with full compatibility with high precision thin needle injection technologies, makes image guided injection, therapeutic planning, interpretation and validation of clinical performance possible. Combined, the unprecedented drug release and imaging properties of CarboCell makes the technology highly attractive for clinical advancement. Citation Format: Sophie B. Jensen, Elizabeth S. Chavez, Hólmfridur R. Haldórsdóttir, Fredrik Melander, Marouschka J. Scheeper, Linda M. Bruun, Jonas R. Henriksen, Anders Elias Hansen, Thomas L. Andresen. CarboCell provides new opportunities for image-guided high-precision intratumoral combinatorial immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1321.

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds.

Surgery is still the first-line treatment for multiple solid cancers. However, recurrence is a common issue, especially when dealing with aggressive tumors or tumors that are difficult to completely remove due to their location. Getting clear surgical margins can be challenging, but treatment strategies combining surgery with other anti-cancer therapies can potentially improve the outcome. Photothermal therapy (PTT) is a technique that relies on photoabsorbing agents, such as gold nanoparticles, to transform light into local hyperthermia. This technique can be used to ablate tumor tissue where the photoabsorbing agent accumulates, sparing healthy surrounding tissue. In this study, we examined the potential of gold nanoparticle-based PTT as an adjuvant treatment to surgery in a mouse model of human fibrosarcoma. For this we performed subtotal tumor resection to mimic a clinical situation where total tumor removal is not achieved, and subsequent PTT was applied on the surgical field. Our results showed that animals undergoing adjuvant PTT after surgery presented sustained delayed tumor growth and improved survival when compared to animals that only underwent surgery. We believe that these findings show the potential of PTT as an adjuvant method to traditional tumor surgery and could pave way to more personalized treatment options.

Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)–sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis.

The main structural element defining the cell is the lipid membrane, which is an integral part of regulating the fluxes of ion and nutrition molecules in and out of the cell. Surprisingly, copper ions were found to have anomalous membrane permeability. This led us to consider a broader spectrum of cations and further a new approach for using liposomes as nanoreactors for synthesis of metal and metal alloy nanoparticles. In the present study, the high membrane permeability of Cu2+ and its neighbouring transition elements in the periodic table was investigated. The permeability of Ni2+, Cu2+, Zn2+, Ag+, Au3+, Mg2+, Ca2+ and Lu3+ was assessed, and we report that Zn2+, Cu2+, Ag+ and Au3+ surprisingly are able to cross lipid bilayers. This knowledge is highly relevant for understanding trafficking of cations in biological systems, as well as for design of novel nanoparticle and nanoreactor systems. An example of its use is presented as a platform for synthesizing single highly uniform gold nanoparticles inside liposomal nanoreactors. We envision that this approach could provide a new nanoreactor methodology for forming highly structurally constrained uniform metal and metal alloy nanoparticles, as well as new methods for in vivo tracking of liposomes.

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging. Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model. Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution. Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 103 antibodies/μm2), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration.

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery.

PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid-loaded functionalized liposomes that exhibit both enhanced uptake by HRECs and superior biologic activity compared to nontargeting liposomes and free drug. METHODS. EPCR expression of HRECs was investigated through flow cytometry and Western blot assays. EPCR-targeting liposomes were developed by functionalizing EPCR-specific antibodies onto liposomes, and the uptake of liposomes was assessed with flow cytometry and confocal laser scanning microscopy. The therapeutic potential of EPCR-targeting liposomes was determined by loading them with prednisolone either through bilayer insertion and/or by remote loading into the aqueous core. The carrier efficacy was assessed in two ways through its ability to inhibit secretion of interleukins in cells stimulated with high glucose and angiogenesis in vitro by using an endothelial cell tube formation assay. RESULTS. HRECs express EPCR at a similar level in both human aortic and umbilic vein endothelial cells. The EPCR-targeting liposomes displayed at least a 3-fold higher uptake compared to nontargeting liposomes. This enhanced uptake was translated into superior anti-inflammatory efficacy, as the corticosteroid-loaded EPCR-targeting liposomes significantly reduced the secretion of IL-8 and IL-6 and inhibited the development of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised.

The enhanced permeability and retention (EPR) effect increases tumor accumulation of liposomal chemotherapy and should, in theory, increase anticancer effects and lower toxicity. Unfortunately, liposomal chemotherapy has generally not met the expected potential, perhaps because the EPR effect is not ubiquitous. PET imaging using radiolabeled liposomes can identify cancers positive for the EPR effect. In the current study, we show in clinical canine cancer patients that repeated imaging with radiolabeled liposomes (64Cu-liposome) induces the accelerated blood clearance (ABC) phenomenon. This was observed even with very long intervals between PEGylated liposome injections, which contradict previous reporting in experimental animal models. The induction of ABC may be devastating for the theranostic use of liposomal imaging, as this could vaccinate patients against therapeutic efficacy. To investigate and solve this important problem, an additional study part was designed in which rats were subjected to repeated liposomal administrations, including stealth 64Cu-liposome PET imaging and Caelyx chemotherapy. Most importantly, it was found that, by increasing the lipid dose at the first injection or by supplying a small predose before the second 64Cu-liposome injection, ABC could be prevented. Importantly, signs of liposome tracer breakdown with subsequent renal excretion were observed. These findings highlight the importance of the ABC phenomenon for liposomal predictive imaging in a clinically relevant setting and show that carefully planned application is central to avoid potential detrimental effects on patient benefit.