Tumor-On-A-Chip Models for Predicting In Vivo Nanoparticle Behavior.

Nanosized drug formulations are broadly explored for the improvement of cancer therapy. Prediction of in vivo nanoparticle (NP) behavior, however, is challenging, given the complexity of the tumor and its microenvironment. Microfluidic tumor-on-a-chip models are gaining popularity for the in vitro testing of nanoparticle targeting under conditions that simulate the 3D tumor (microenvironment). In this review, following a description of the tumor microenvironment (TME), the state of the art regarding tumor-on-a-chip models for investigating nanoparticle delivery to solid tumors is summarized. The models are classified based on the degree of compartmentalization (single/multi-compartment) and cell composition (tumor only/tumor microenvironment). The physiological relevance of the models is critically evaluated. Overall, microfluidic tumor-on-a-chip models greatly improve the simulation of the TME in comparison to 2D tissue cultures and static 3D spheroid models and contribute to the understanding of nanoparticle behavior. Interestingly, two interrelated aspects have received little attention so far which are the presence and potential impact of a protein corona as well as nanoparticle uptake through phagocytosing cells. A better understanding of their relevance for the predictive capacity of tumor-on-a-chip systems and development of best practices will be a next step for the further refinement of advanced in vitro tumor models.

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns-/- larvae, and restoration of the zebrafish pronephros function.

Deciphering Structural Determinants Distinguishing Active from Inactive Cell-Penetrating Peptides for Cytosolic mRNA Delivery.

The formation of noncovalent complexes by mixing of positively charged polymers with negatively charged oligonucleotides (ONs) is a widely explored concept in nanomedicine to achieve cellular delivery of ONs. Uptake of ON complexes occurs through endocytosis, which then requires release of ON from endosomes. As one type of polymer, cell-penetrating peptides (CPPs) are being used which are peptides of about 8-30 amino acids in length. However, only a few CPPs yield effective cytosolic ON delivery and activity. Several strategies have been devised to increase cellular uptake and enhance endosomal release, among which an increase of osmotic pressure through the so-called proton sponge effect, disruption of membrane integrity through membrane activity, and disulfide-mediated polymerization. Here, we address the relevance of these concepts for mRNA delivery by incorporating structural features into the human lactoferrin-derived CPP, which shows uptake but not delivery. The incorporation of histidines was explored to address osmotic pressure and structural motifs of the delivery-active CPP PepFect14 (PF14) to address membrane disturbance, and finally, the impact of polymerization was explored. Whereas oligomerization increased the stability of polyplexes against heparin-induced decomplexation, neither this approach nor the incorporation of histidine residues to promote a proton-sponge effect yielded activity. Also, the replacement of arginine residues with lysine or ornithine residues, as in PF14, was without effect, even though all polyplexes showed cellular uptake. Ultimately, sufficient activity could only be achieved by transferring amphipathic sequence motifs from PF14 into the hLF context with some benefit of oligomerization demonstrating overarching principles of delivery for CPPs, lipid nanoparticles, and other types of delivery polymers.

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA.

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.

Block or degrade? Balancing on- and off-target effects of antisense strategies against transcripts with expanded triplet repeats in DM1.

Antisense oligonucleotide (ASO) therapies for myotonic dystrophy type 1 (DM1) are based on elimination of transcripts containing an expanded repeat or inhibition of sequestration of RNA-binding proteins. This activity is achievable by both degradation of expanded transcripts and steric hindrance, although it is unknown which approach is superior. We compared blocking ASOs with RNase H-recruiting gapmers of equivalent chemistries. Two DMPK target sequences were selected: the triplet repeat and a unique sequence upstream thereof. We assessed ASO effects on transcript levels, ribonucleoprotein foci and disease-associated missplicing, and performed RNA sequencing to investigate on- and off-target effects. Both gapmers and the repeat blocker led to significant DMPK knockdown and a reduction in (CUG)exp foci. However, the repeat blocker was more effective in MBNL1 protein displacement and had superior efficiency in splicing correction at the tested dose of 100 nM. By comparison, on a transcriptome level, the blocking ASO had the fewest off-target effects. In particular, the off-target profile of the repeat gapmer asks for cautious consideration in further therapeutic development. Altogether, our study demonstrates the importance of evaluating both on-target and downstream effects of ASOs in a DM1 context, and provides guiding principles for safe and effective targeting of toxic transcripts.

Light-Responsive Elastin-Like Peptide-Based Targeted Nanoparticles for Enhanced Spheroid Penetration.

We describe here a near infrared light-responsive elastin-like peptide (ELP)-based targeted nanoparticle (NP) that can rapidly switch its size from 120 to 25 nm upon photo-irradiation. Interestingly, the targeting function, which is crucial for effective cargo delivery, is preserved after transformation. The NPs are assembled from (targeted) diblock ELP micelles encapsulating photosensitizer TT1-monoblock ELP conjugates. Methionine residues in this monoblock are photo-oxidized by singlet oxygen generated from TT1, turning the ELPs hydrophilic and thus trigger NP dissociation. Phenylalanine residues from the diblocks then interact with TT1 via π-π stacking, inducing the re-formation of smaller NPs. Due to their small size and targeting function, the NPs penetrate deeper in spheroids and kill cancer cells more efficiently compared to the larger ones. This work could contribute to the design of "smart" nanomedicines with deeper penetration capacity for effective anticancer therapies.

Co-Culture of Glomerular Endothelial Cells and Podocytes in a Custom-Designed Glomerulus-on-a-Chip Model Improves the Filtration Barrier Integrity and Affects the Glomerular Cell Phenotype.

Crosstalk between glomerular endothelial cells and glomerular epithelial cells (podocytes) is increasingly becoming apparent as a crucial mechanism to maintain the integrity of the glomerular filtration barrier. However, in vitro studies directly investigating the effect of this crosstalk on the glomerular filtration barrier are scarce because of the lack of suitable experimental models. Therefore, we developed a custom-made glomerulus-on-a-chip model recapitulating the glomerular filtration barrier, in which we investigated the effects of co-culture of glomerular endothelial cells and podocytes on filtration barrier function and the phenotype of these respective cell types. The custom-made glomerulus-on-a-chip model was designed using soft lithography. The chip consisted of two parallel microfluidic channels separated by a semi-permeable polycarbonate membrane. The glycocalyx was visualized by wheat germ agglutinin staining and the barrier integrity of the glomerulus-on-a-chip model was determined by measuring the transport rate of fluorescently labelled dextran from the top to the bottom channel. The effect of crosstalk on the transcriptome of glomerular endothelial cells and podocytes was investigated via RNA-sequencing. Glomerular endothelial cells and podocytes were successfully cultured on opposite sides of the membrane in our glomerulus-on-a-chip model using a polydopamine and collagen A double coating. Barrier integrity of the chip model was significantly improved when glomerular endothelial cells were co-cultured with podocytes compared to monocultures of either glomerular endothelial cells or podocytes. Co-culture enlarged the surface area of podocyte foot processes and increased the thickness of the glycocalyx. RNA-sequencing analysis revealed the regulation of cellular pathways involved in cellular differentiation and cellular adhesion as a result of the interaction between glomerular endothelial cells and podocytes. We present a novel custom-made glomerulus-on-a-chip co-culture model and demonstrated for the first time using a glomerulus-on-a-chip model that co-culture affects the morphology and transcriptional phenotype of glomerular endothelial cells and podocytes. Moreover, we showed that co-culture improves barrier function as a relevant functional readout for clinical translation. This model can be used in future studies to investigate specific glomerular paracrine pathways and unravel the role of glomerular crosstalk in glomerular (patho) physiology.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

The potential of RNA-based therapy for kidney diseases.

Inherited kidney diseases (IKDs) are a large group of disorders affecting different nephron segments, many of which progress towards kidney failure due to the absence of curative therapies. With the current advances in genetic testing, the understanding of the molecular basis and pathophysiology of these disorders is increasing and reveals new potential therapeutic targets. RNA has revolutionized the world of molecular therapy and RNA-based therapeutics have started to emerge in the kidney field. To apply these therapies for inherited kidney disorders, several aspects require attention. First, the mRNA must be combined with a delivery vehicle that protects the oligonucleotides from degradation in the blood stream. Several types of delivery vehicles have been investigated, including lipid-based, peptide-based, and polymer-based ones. Currently, lipid nanoparticles are the most frequently used formulation for systemic siRNA and mRNA delivery. Second, while the glomerulus and tubules can be reached by charge- and/or size-selectivity, delivery vehicles can also be equipped with antibodies, antibody fragments, targeting peptides, carbohydrates or small molecules to actively target receptors on the proximal tubule epithelial cells, podocytes, mesangial cells or the glomerular endothelium. Furthermore, local injection strategies can circumvent the sequestration of RNA formulations in the liver and physical triggers can also enhance kidney-specific uptake. In this review, we provide an overview of current and potential future RNA-based therapies and targeting strategies that are in development for kidney diseases, with particular interest in inherited kidney disorders.

Gelatin Nanoparticles for Complexation and Enhanced Cellular Delivery of mRNA.

Messenger RNA (mRNA) is increasingly gaining interest as a modality in vaccination and protein replacement therapy. In regenerative medicine, the mRNA-mediated expression of growth factors has shown promising results. In contrast to protein delivery, successful mRNA delivery requires a vector to induce cellular uptake and subsequent endosomal escape to reach its end destination, the ribosome. Current non-viral vectors such as lipid- or polymer-based nanoparticles have been successfully used to express mRNA-encoded proteins. However, to advance the use of mRNA in regenerative medicine, it is required to assess the compatibility of mRNA with biomaterials that are typically applied in this field. Herein, we investigated the complexation, cellular uptake and maintenance of the integrity of mRNA complexed with gelatin nanoparticles (GNPs). To this end, GNPs with positive, neutral or negative surface charge were synthesized to assess their ability to bind and transport mRNA into cells. Positively charged GNPs exhibited the highest binding affinity and transported substantial amounts of mRNA into pre-osteoblastic cells, as assessed by confocal microscopy using fluorescently labeled mRNA. Furthermore, the GNP-bound mRNA remained stable. However, no expression of mRNA-encoded protein was detected, which is likely related to insufficient endosomal escape and/or mRNA release from the GNPs. Our results indicate that gelatin-based nanomaterials interact with mRNA in a charge-dependent manner and also mediate cellular uptake. These results create the basis for the incorporation of further functionality to yield endosomal release.

Biomaterial-Mediated Protein Expression Induced by Peptide-mRNA Nanoparticles Embedded in Lyophilized Collagen Scaffolds.

In our aging society, the number of patients suffering from poorly healing bone defects increases. Bone morphogenetic proteins (BMPs) are used in the clinic to promote bone regeneration. However, poor control of BMP delivery and thus activity necessitates high doses, resulting in adverse effects and increased costs. It has been demonstrated that messenger RNA (mRNA) provides a superior alternative to protein delivery due to local uptake and prolonged expression restricted to the site of action. Here, we present the development of porous collagen scaffolds incorporating peptide-mRNA nanoparticles (NPs). Nanoparticles were generated by simply mixing aqueous solutions of the cationic cell-penetrating peptide PepFect14 (PF14) and mRNA. Peptide-mRNA complexes were uniformly distributed throughout the scaffolds, and matrices fully preserved cell attachment and viability. There was a clear dependence of protein expression on the incorporated amount of mRNA. Importantly, after lyophilization, the mRNA formulation in the collagen scaffolds retained activity also at 4 °C over two weeks. Overall, our results demonstrate that collagen scaffolds incorporating peptide-mRNA complexes hold promise as off-the-shelf functional biomaterials for applications in regenerative medicine and constitute a viable alternative to lipid-based mRNA formulations.

Association of MMP-2 genes variants with diabetic retinopathy in Tunisian population with type 2 diabetes.

AIMS: Few studies investigated the association of genetic difference in metalloproteinase-2 (MMP-2) gene with diabetic retinopathy but with mixed outcome. To investigate the association between a set of MMP-2 genetic variants and the risk of diabetic retinopathy in an Arab Tunisian population with type 2 diabetes. SUBJECTS AND METHODS: A retrospective case-control study comprising a total of 779 type 2 diabetes patients with or without diabetic retinopathy was conducted. Genotyping was prepared by TaqMan® SNP genotyping qRT-PCR. The variants used were rs243865 (C/T), rs243864 (T/G), rs243866 (G/T) and rs2285053 (C/T). RESULTS: The minor allele frequency (MAF) of the rs243864 MMP-2 variant was significantly higher among diabetic retinopathy patients. Setting homozygous wild type genotype carrier as reference, the rs243864T/G allele was associated with increased risk of diabetic retinopathy under the dominant, recessive, and additive models which persisted when key covariates were controlled for, while a reduced risk of diabetic retinopathy progression was seen after adjustment between non-proliferative and proliferative diabetic patients. Furthermore, the heterozygous genotype GT of the rs243866 variant is positively associated with the risk of proliferative diabetic retinopathy in the additive model. A limited linkage disequilibrium (LD) was revealed between the four-matrix metalloproteinase-2 variants. Four-loci haplotype analysis identified, GCTC, TTTC, and GCTT haplotypes to be positively associated with the risk of diabetic retinopathy. CONCLUSION: Our findings demonstrate that the MMP-2 variant rs243864 and 243866 are related to the susceptibility to diabetic retinopathy and the progression of the disease in an Arab Tunisian population with type 2 diabetes.

Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen.

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.

Delivery of antisense oligonucleotides for splice-correction of androgen receptor pre-mRNA in castration-resistant prostate cancer models using cell-penetrating peptides.

BACKGROUND: Cell-penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre-mRNA, thereby preventing the generation of the splice variant AR-V7 mRNA. AON-mediated knockdown of AR-V7 resulted in inhibition of androgen-independent cell proliferation. In this study, we evaluated the CPP-mediated delivery of this AON into castration-resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). METHODS: Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter-Glo cell viability assay. The effectivity of CPP-mediated delivery of the splice-correcting AON-intronic splicing enhancer (ISE) targeting the ISE in the castration-resistant prostate cancer (CRPC)-derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR-V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR-V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. RESULTS: The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine-rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier-associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 µM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON-ISE directed against AR pre-mRNA achieved significant downregulation of AR-V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen-independent cell proliferation of DuCaP and VCaP cells. CONCLUSIONS: This proof-of-principle study constitutes the basis for further development of CPP-mediated delivery of AONs for targeted therapy in prostate cancer.

Imaging of CPP Delivery Mechanisms of Oligonucleotides.

Cationic cell-penetrating peptides spontaneously associate with negatively charged oligonucleotides to form submicron nanoparticles, so-called polyplexes. Contact with cells leads to endosomal uptake of these nanoparticles. Oligonucleotide activity critically depends on endosomal release and finally dissociation of polyplexes. Fluorescence provides a highly powerful means to follow the spatial dynamics of oligonucleotide uptake, trafficking and decomplexation, in particular when combined with markers of subcellular compartments that enable a quantitative analysis of colocalization and thereby mapping of trafficking routes. In this chapter, we describe protocols for a highly defined formation of polyplexes. We then point out the use of fluorescent fusion proteins to identify subcellular trafficking compartments and image analysis protocols to obtain quantitative information on trafficking routes and endosomal release.

Constrained peptides mimic a viral suppressor of RNA silencing.

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.

Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform.

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system's feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application.

CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody.

Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.

Protein Expression Correlates Linearly with mRNA Dose over Up to Five Orders of Magnitude In Vitro and In Vivo.

Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose-response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipid-based formulation and the cell-penetrating peptide Pepfect14 (PF14) were employed. As cell lines, we included a glomerular endothelial cell line (mGEnC) as a model for differentiated cells, HeLa cells, and SKOV-3 ovarian carcinoma cells. Uptake and expression were detected by flow cytometry, using a Cy5-labelled mRNA coding for enhanced green fluorescent protein (EGFP). There was a linear correlation of dose, uptake, and expression, and this correlation was maintained for over up to 72 h. Through application of a multistep kinetic model, we show that differences in expression levels can already be explained by the number of mRNAs packaged per delivery vehicle. Using luciferase as a reporter protein, linearity of expression was observed over 5 orders of magnitude in vitro and 3 orders of magnitude in vivo. Overall, the results demonstrate that mRNA provides excellent quantitative control over protein expression, also over extended periods of time.

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy.

The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.