Nanomedicines: selective targeting, hybrid systems and combined therapy

Nanomedicines: selective targeting, hybrid systems and combined therapy

Professor Mafalda Ascensão Videira describes her group’s work on developing nanomedicines for cancer treatments, and describes some of the main barriers to nanomedicine.

Considerable expertise and public funding have been dedicated to ‘nano’ scale therapeutics, generating a myriad of new ideas and entrepreneur opportunities. As a result, abundant evidence data on nanomedicines is now available for the scientific community. However, are they prepared to fight cancer heterogeneity? Considering the size scale and the consequent ratio of surface area to volume, pharmaceutical nanomedicines may well fight several cell aberrations, interact with a multitude of cell types and suppressing traditional body barriers.

Nanomedicines may be defined as a broad range of pharmaceutical tools that can be used successfully to delivery therapeutic agents or to be used for disease diagnosis or prevention. Those are unique systems that by stepping in at the intersection of materials physics and molecular biology have contributed to a better understanding of the complexity underlying the pathophysiology of several diseases.

The nanoparticles (Np) developed by our group comprise configurable pharmaceutical delivery systems ranging from 10 to 250nm, designed to shield molecules or convey them at its surface thus solving drug or biologic molecule limits related to stability, toxicity, unwanted tissue distribution profile or difficulties involved in overcoming biological barriers. This work intends to build up a synopsis on the delivery strategies currently developed by our group, mainly those dedicated to eradicating cancer.

Reposition of conventional drugs with new levels of pharmacological efficiencies
Disappointing clinical outcomes in cancer therapeutic management are associated to multidrug resistance (MDR), whereby cancer cells become resistant to the cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents. A group of protein cell membranes (MDR) mediate cell membrane drug transport, cytosolic delivery and efflux. Drug efflux is an event that severely limits a drug’s effectiveness. Frequently involving cell’s P-gp overexpression, it could both be responsible for the low pharmacologic action in situ as well as for increased adverse reactions related to unspecific drug-tissue targeting.

This frustrating dose-limiting situation have inspired and driven our group scientific goal: engineering nanomedicines to bridging intracellular delivery of therapeutics.
To this end, we initiate a stepwise development and process optimisation of innovative Np using biomaterials such as, lipids, polymers or both to encapsulate paclitaxel (PTX) as model drug.

At this point we have distinguished two types of outcomes: the impact in cell viability and the ability to evade the membrane protein Pg-P. Furthermore, while the magnitude of the effect may differ from cell to cell, we hypostasised that if drug encapsulation has implications on their cellular accumulation, it will also affect Pg-p ability to recognise its substrate: the drug.

Breast (MCF-7, MDA-MB-231, MDA-MB-468 and SKBR3), colon (HCT8 and HCT116), glioblastoma (U87MG) and lung (A549), have been used to test the free drug activity and compare it with the effect on cell viability and Pg-p membrane expression in cells upon incubation with encapsulated drug. Our data undoubtedly illustrates that the free drug needs to be transported to the cytosol, justifying the observed increase in cell membrane Pg-p expression.1 No matter the biomaterial used to encapsulate the drug, Np cellular uptake by endocytosis avoided recognition and further efflux of the drug, increased drug intracellular accumulation and bioavailability rendering a decrease on cell viability.
Owing nanomedicines capability to encapsulate a broad range of clinically relevant agents, drug reposition trough nanomedicines could be a reality.

Interference with cell malignancy using new engineered therapeutics
Perhaps a major paradigm change in oncology has taken place with the findings in genomics and post-genomics and its implementation as a research tool. Looking at the cell as a molecular machine has had an impact in cancer research providing a more knowledge-based approach to select the right target. As such, tumour classification can be seen as the imbalance between regulated and unregulated cells and classified according to the level of irregularity; genome, transcriptome, proteome or metabolome cell disease. Information gathered so far has allowed the scientific community to ‘pin’ gene therapy as a truly pharmaceutical option.

We engage in this opportunity by loocking out for phenotypic regulations at the post-transcriptional level as potential new targets for cancer eradication — post-transcriptional effectors like small interfering RNA (siRNA) for protein/gene down-regulation. Efficacy depends on its cytosolic availability to initiate gene silencing: translocation of the artificially designed siRNA across the target cell membrane and subsequent trafficking and/or release of siRNA into the cytoplasm.

From our point of view, nanomedicines are the best strategy to attain what we call the ‘rise of silence’. By 2010, we took the risk of using nanomedicines to validate the silencing of the Akt2 protein as an anticancer target. Besides being a survival protein, the Akt2 involvement in acquisition of stem cell-like properties which are responsible for invasiveness and chemo-resistance, promoted by the transcriptional factor Twist —the cell ‘hub’ – involved in acquisition of a mesenchymal phenotype through Epithelial-Mesenchymal Transition (EMT).

Anticipating a great deal of innovation, the project ‘siRNA incorporation in a lipid/micelle-based nanocarrier as a strategy to restore E-cadherin expression and eradicate advanced breast cancer metastatic phenotype’ receives funding from the Portuguese Science Foundation (PTDC/SAU-FAR/120453/2010).

Overall, the data obtained allowed us to clarify that the Akt2 down-regulation correlates with intracellular accumulation of AKT2siRNA encapsulated in nanomedicines upon endocytosis. The cytosolic availability further affects negatively the AKT2/Twist axis which does triggers E-cadh restoration, with a subsequent restrain of tumourigenic phenotype observed by the decrease in the stemness proteins, such as Vimentine (see Fig 1).2
Proteins down regulation by blocking mRNA translation is intimately related to the sequence-specific siRNA ability to interact accurately (Purchased available). Notably, from a pharmaceutical perspective, the observed in vitro and in vivo biological activity highlights this polymeric-based nanomedicine ability to overcome both extracellular and intracellular barriers delivering the intact siRNA sequence in the cell cytosol.3

Evaluation of the potential for antibody-guided selective targeting

Another challenge surrounding conventional clinical management is related to the inability of the administered drugs to discriminate between tumour and normal cells, thereby urging the recognised systemic toxicity and adverse events. Targeting selectively the injury tissue/cell should avoid these limitations though retaining the drug in the desired body region.

Passive targeting and tumour accumulation of nanomedicines is a key point that takes advantage of the injury tissue leak vasculature i.e., defective vessels and impaired lymphatic drainage, a phenomenon known as the enhanced permeability and retention.4 However, in advanced stages, highly aggressive tumour subpopulations survive! To do that, they express a massive amount of membrane receptors to attain their own proliferation, cell renewal capacity (cancer stem cells) and interaction with the tumour microenvironment.

These seek the attention of a multidisciplinary team, namely by using the membrane receptor CD44, one of the most predominant stemness markers found in diverse phenotypes associated with tumour progression. Understandably, a consequence of the nanoparticles low mean diameter is the high surface area available to bond target moieties. Polymer-based colloidal dispersions, composed of amphiphilic molecules PLGA-co-PEG-Paclitaxel which self-assemble to form paclitaxel loaded nanomedicines were subsequently decorated with a specific ligand with affinity for the CD44 receptor (CD44v6). By doing that, we prove that CD44 surface decoration improves nanomedicines efficacy suggesting that active targeting increases the sensitivity of CSC to the loaded PTX .5

Remarkably, cell viability and tumoursphere formation capacities are significantly lower when PTX is delivered in CD44 decorated nanomedicines. On the contrary, the percentage of CSC within all tested tumour cell lines increase after treatment with PTX as well as upon incubation with non-functionalised PLGA-co-PEG-PTX. European agencies respond positively by funding a project ENMed/0009/2015 (Traget4Cancer) that already renders more selective and accurate therapeutically agents.

The already-created network of experts in nanotechnology, cytotoxicity assays, cancer cell testing, tumour cell animal models and statistical analysis have made it possible to build a complex experimental design aiming to move on to a multiple targets’ strategy – co-delivering siRNA alongside with the specific siRNA against Akt2 paclitaxel combined with the antibody-guided approach. To this end, CXCR4 and EGFR6 CSC expressed cell surface markers were targeted throughout the surface functionalisation with CXCR4 antagonist peptide (Peptide R) or EGFR antibodies (Cetuximab). EGFR chimeric monoclonal antibody, Cetuximab are currently used for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer and head and neck cancer.

Not surprisingly, considering the relevance of the nanomedicine’s physicochemical properties, the optimal and stable dosage form with two different types of molecules shielded into the same nanoparticle as proved to be a gigantic task that is yet to be completed. Nevertheless, physical mixtures of each system are being tested in vitro. Data from immunocytochemical analysis confirms that both systems co-exist in the cells cytosol. It is up to us to decide if this could be considered a mitigation solution. From our project’s data, we can already conclude that a convergence amongst nanomedicines, siRNAi efficacy and antibody-guided strategy is a potent tool against the marginal highly malignant CSC subpopulations.7

Gathering momentum

Moving forward from our status quo at that point, the group took an initiative denominated ‘framing all the picture’ designed to attack multiple targets in different disease stages in an extremely synchronised way. We still don’t know at what point one or more cell molecular pathways conquer tumourigenesis control at the growth, survival and dissemination level. But our data has shown that targeting cell receptors using available therapeutic antibodies for selective NP delivery achieved effective Drug-Np intracellular accessibility together with siRNA-Np’s capable of revert cells phenotype is possible.

As such, the current research project ENMed/0065/2016 (NanoGlio) was envisioned keeping in mind that modulators involved in circulating tumour cells (CTC) genomics and proteins that support CTC chemiotaxis to the brain can be therapeutic targets. Pharmaceutical Np proposed to this end are hybrid functionalised systems loaded with therapeutics/immunomodulators. Experimental data from in vitro/in vivo using models of murine glioma (GBM) will show its efficacy for brain delivery, tumour abrogation and defeat tumour immunosuppressive properties.

Diffusing into the tumour against hydrostatic pressure to achieve therapeutic effectiveness in this exclusive biochemical and immunological niche is a challenge. Prior to this, pharmaceuticals have had to cross the impermeable Blood Brain Barrier (BBB).8 Recent seminal work has provided compelling evidence that RVG, a small peptide derived from the rabbies (rb) capsid, promotes transvascular delivery to the central nervous by receptor-mediated transcytosis upon association with the acetylcholine receptor (AchR), widely expressed in the brain, including endothelial cells. Its clinical relevance and potential applicability grab our attention as it may act as targeting moiety of the Np herein design.

Although active targeting could be a more complex biological interaction, nanomedicines surface decoration with RVG to achieve siRNA-Np-Drug accumulation in brain, knock-down Akt2 expression and further decreasing mesenchymal markers is our group’s end goal.
At this stage, the stemness proteins used as biomarkers to identify cell phenotype and dissemination potential of cells are, Akt2, Vimentin, TWIST; CD44; E-cadherin, ZEB 1 and 2. High-throughput screening of aberrantly expressed miRNA, already accomplished by Professor Alexandra Brito (iMed), brought to light the crucial role of this nucleotides as ‘information-conveyers’ , also providing a broad range of markers that might be significant to trace the tumourigenesis and hopefully preventing it.

‘Framing all the picture’ starts as a provoking concept that would be achieved if our approach could selectively interfere with several signalling pathways that regulate proliferation, survival and abnormal cell functions, which is a smart approach against tumour heterogeneity.

Our previously gathered information about combination therapies, hybrid nanomedicines, antibody-guided targeting underlies the ambition of achieving tumour microenvironment infiltration, maximising the chances of clinical complete brain tumour or breast metastasis eradication e.g., primary region and distant metastasis. We aim also to block the circulation of tumour cells and impair premetastatic chemotaxis. To assure brain metastasis selective targeting, a dual system drug co-encapsulated with a RNAi-poliplexes was designed against of cells with different phenotypes, thus eradicating heterogeneous cancer types typically present in disseminated invasive disease. A platform to achieve breast cancer brain metastasis eradication is an ambitious goal that was funded recently by FCT (PTDC/MED-ONC/29402/2017).

The procedure for constructing the correct BBB-drug delivery system as nanomedicine is time, resource and cost intensive. Thus, in silico screening/filtering and optimising that step would assist the experimental work in all phases and is now being used. The expected upcoming outcomes might, from our point of view, be transversal to other metastisation mechanisms open a new era to fight advanced tumours.

Exploring alternative administration routes

Between 2004 and 2012, our group has consistently proven that the developed Paclitaxel-loaded Solid Lipid Nanoparticles (SLN) succeeded in the total suppression of the lung metastases through the lymphatic system after pulmonary administration.8 Formerly, reaching the tumour mass and undergoing cell internalisation through the systemic circulation can only be devised by parenteral administration. We went to another level; using the lung as a local administration route reaching the lymphatic system upon nanomedicines lung deposition. A patented SLN technology meant that we were able to incorporate lipophilic molecules, such as PTX, under mild conditions, avoiding harsh stress conditions during production.10

Until now, all developed nanomedicines or combinations thereof are submitted to a multifactorial design of experiments approach, based on the ICH Pharmaceutical Development guidelines (ICH Q8 (R2)) (ICHQ8(R2), 2009). The resulting pharmaceutical product must present a set of quality attributes that will define the target quality product profile (QTPP). As such, we started by defining some acceptance limits – mean size (MS) lower than 250nm, a polydispersity index (PI) lower than 0.250, a drug entrapment efficiency (EE%) higher than 90% and the absence of significant changes in these parameters after storage at room temperature up to six months. Systems developed by our group are mostly envisioned for pulmonary administration even though intravenous administration is also used.

Regulatory issues

The propensity to adopt and regulate innovation requires knowledge. Nanomedicines can be designed with different sizes, shapes and surface properties, thus presenting different pharmacological and biological properties since body pharmacokinetics are tightly related to the designed nanostructure.

Authorisation to get to the market demands massive physicochemical and biological information which are based on standardised and reliable methodologies. The combination of computer-based molecular modelling and the parallel assessment of biological properties by robust experimental methodology and statistical analysis can open new avenues in this field of scientific research by providing powerful tools for rationale design and optimisation of this nanotechnology-based drug delivery systems.

Among others, consistency in the manufacturing process, namely in terms of nanomedicine’s mean size, drug concentration and formulation polydispersity index, are all considered as being critical quality attributes once they ultimately limit the administration route, as required by pharmacopoeias and regulatory agencies. A way to circumvent the uncertainty would be by taking advantage of the pharmaceutical industry’s installed capabilities to decrease the overall costs of scaling-up, while increasing regulatory compliance and flexibility.

RNAi technology acceptance, though mediated by pharmaceutical nanomedicines, faces the paradox of interfering with the human biological environment. Due to the fact that all cell physiological processes can be target candidates, silencing a precise biological pathway could be challenging if target selectivity is not properly addressed. Fortunately, molecular biology has provided robust scientific tools to suppress some of the most critical issues in gene therapy, while setting the standards for siRNA clinical application.
Review testing methodologies evaluating the adequacy of theb in vivo pre-clinical models and identifying synergies has been our group commitment and an undergoing task that takes place under a COST Action (MP1404, ‘SimInahle’).11

Conclusion

The balance between the nanomedicines therapeutic add value and the human safety factor poses enormous ethical issue. Most knowledge gained from exploratory early translational research does not fit in the conventional preclinical and/or clinical programs. From the regulator’s point of view, a roadmap agreement towards common quality target product profile (QTPP) attributes as well as manufacture processes based on the ICH Pharmaceutical Development guidelines (ICHQ8(R2), 2009) would lead to well structured, potentially more simplified development programmes.

Lack of consistency may, in fact, be the strongest barrier to accept the new categories of pre-clinical evidence and consequently achieve the nanomedicines full potential in clinical use.

To enable nanomedicines to reach the patient, this field must have robust predictive tools and the thorough understanding of the physical and chemical properties of the materials at this scale. Improving their quality, consistency and manufacturability could be the missing step that would ultimately lead to the construction of versatile platforms that can be useful for a range of severe diseases or, more important, to fulfil unmet clinical needs.

Finally, industry’s decline in expenditure especially in therapeutically or vaccine- based nanomedicines, as frightening as it is, could be an opportunity for partnerships with academia pushing forward research opportunities and pushing the integration between different players and making good use of the financial support, both public or private. Funding agencies are enthusiastic about the results obtained so far, including their contribution to the risk share model that is crucial to achieving intersections and igniting the necessary risk taking on the part of big pharma.

Considering that any given drug, or biological molecule, could be encapsulated in a nanomedicine, one could speculate that the number of available combinations would be unlimited.

Acknowledgements
Professor Beatriz Silva Lima, Head of the Pharmacological and Regulatory Sciences
Group (PharmaRegSci).
Fundação para a Ciência e a Tecnologia, Portugal (FCT) (PTDC/SAU- FAR/120453/2010; PTDC/MED-ONC/29402/2017), iMed.ULisboa (UID/DTP/04138/2013).
EuroNanonet ( ENMed/0009/2015 – Traget4Cancer; ENMed/0065/2016 – NanoGlio).

References
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3 Rafael D, Doktorovová S, Florindo H.F, Gener P, Abasolo I, Schwartz S.Jr, Videira MA (2015). ‘EMT blockage strategies: targeting Akt dependent mechanisms for breast cancer metastatic behaviour modulation’. Current Gene Therapy. 15 (3): 300-12;
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Mafalda Ascensão Videira
Faculdade de Farmácia da Universidade de Lisboa
+ 35 (1) 919 539 400
mafaldavideira@campus.ul.pt
Tweet @iMedULisboa
http://imed.ulisboa.pt/cv/mafalda-marques-videira/

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