The blood brain barrier (BBB) is a highly specialized network of blood vessels that effectively separates the brain environment from the circulatory system. Delivering drugs into the brain environment has historically been a challenge. This is due to the nature of the BBB anatomy, as it is composed of polarized endothelial cells that are connected by tight junctions of the cerebral capillary endothelium and a variety of transporters (Figure 1). As a result of this, the treatment for brain diseases such as primary or secondary brain tumours, Alzheimer’s and multiple sclerosis remains complicated.

Figure 1. Summary of BBB anatomy and transport pathways (Adapted from Bukari et al., 2020).

Currently, various non-invasive strategies have been developed for treating the brain disease such as focused ultrasound, nanoparticle and antibody mediated delivery, however, they still come with risks and limitations. Moreover, antibodies, capable of binding to multiple protein targets pose a serious risk of eliciting an immune response that could damage brain tissues and organs that are otherwise healthy. Therefore, there is a need to develop methods of delivering therapeutics efficiently, while minimizing risks of adverse effects.

Aptamer: The Antibody Alternative:

Aptamers, owing to the characteristics they possess, have shown promising preliminary results for transporting therapeutics into the brain. Advantages of aptamers include:

  • Immunologically inert, which means they are less likely to instigate an immune reaction when introduced to the brain environment. This is the most important feature, especially since immunogenic reactions in the brain environment could negatively affect healthy tissues in the central nervous system (CNS), which could lead to serious complications.
  • Small size (30 kDa for a 100 bp ssDNA) enables them to penetrate cells and tissues of the brain more efficiently.
  • Chemically modifiable to serve improving binding affinity, stability and functionalize them via conjugation to reporter molecules or therapeutic cargos. This strategy allows for the development of targeted delivery of drugs.
  • Easily customizable to suit various required assays for effective diagnosis and treatment.
  • No batch-to-batch variation as they are synthesized at high fidelity via oligonucleotide synthesis process, making for a more consistent end product.

Overall, being small, modifiable, and non-immunogenic molecules allows for the development of more efficient and drug transport strategies that can overcome the challenges of passing through the BBB. Considering these properties of aptamers, several research groups have developed aptamers that can target the BBB endothelial cells (EC) or proteins on its membrane to negotiate a way across the BBB. We review two of such promising findings here that can be effective in future glioma and tauopathy treatment.

Aptamers crossing BBB for effective glioma therapy

Glioblastoma multiforme (GBM) or glioma is a frequent type of aggressive primary brain tumour associated with a high mortality rate and poor prognosis. Effective treatment for brain glioma needs to conquer two barriers: the BBB and the brain glioma barrier. Most of the chemotherapies alone have failed because of the blood brain barrier resistance and poor glioma targeting of the chemotherapeutics.

Dual or multiple targeting systems have been identified as one of the most promising therapies. Shi et al., 2019 established a multifunctional nanosystem for targeting glioma. In this study, the author utilized a novel nano-carrier, 3D tetrahedral framework nucleic acid (tFNA) to carry two established and functionalized aptamers GMT8 and Gin4.T forming a network – Gint4.T – tFNA – GMT8 (GTG). This novel aptamer network GTG could target U87MG cells and inhibit the proliferation of glioma. GTG was efficiently internalized by the target U87MG and bEnd.3 cells and penetrated an in-vitro blood brain barrier model (Figure 2).

Figure 2: GTG could pass an in vitro BBB model. U87MG cells were seeded in the low trans well compartment. Cy5 labeled tFNA and GTG were added to the upper chamber to assess the penetration efficiency of tFNA and GTG across the BBB. Confocal microscopy showed significant uptake of GTG in the cytoplasm of U87MG cells as compared to tFNA alone.

GTG loaded with paclitaxel (GPC) showed anti-glioma efficacy. It inhibited the proliferation, migration and invasion of U87MG cells and enhanced apoptosis induction in these cells (Figure 3). The study overall demonstrated aptamer mediated paclitaxel delivery had great potential for glioma treatment.

Figure 3. A,B,C,D – U87MG cells treated with GTG, PTX, and GPC were subjected to flow cytometry and confocal microscopy  to detect apoptosis. E. Migration experiment on U87MG cells treated with GTG, PTX, and GPC showed that the inhibitory effect of GPC on U87MG cells was significantly stronger than GTG and PTX alone.

Aptamer crossing BBB for effective Tauopathy therapy

Tauopathies, including Alzheimer’s disease, Dementia and Parkinsonism are a group of neurodegenerative diseases characterized by the abnormal deposition of hyper phosphorylated Tau (P-Tau) filaments. So far, only limited success has been achieved in Tau based therapeutic interventions for tauopathies. In this view, a very recent article by Li et al., 2020 reported the synthesis of a circular bifunctional aptamer to enhance the in vivo BBB penetration for better tauopathy therapy.

The circular aptamer consisted of two highly functional aptamers:

  • Transferrin receptor (TfR) aptamer to facilitate TfR – aptamer recognition – induced transcytosis across BBB endothelial cells
  • Tau protein aptamer that could inhibit Tau phosphorylation and other tauopathy – related pathological events in the brain (Figure 4).

The targeting capability of the circular aptamer was evaluated both in vitro and in vivo.

Figure 4. Top image – A. Schematic representation of formation of circular Tau and transferrin receptor (TfR) bispecific aptamer via enzymatic ligation. B. Transport of circular aptamer crossing BBB via the specific recognition between TfR and TfR aptamer and its potential disruption of tauopathy at several points. C. Agarose gel analysis of the circular aptamer and its components. Bottom image – In vitro targeting capability of the circular aptamer against the target bEnd 3 cells confirmed by confocal microscopy and flow cytometry.

This novel modified circular aptamer displayed enhanced plasma stability, brain exposure and improved BBB permeability in both in vitro and in vivo studies. Most importantly, the circular Tau-TfR bispecific aptamer led to attenuated brain levels of Tau with the ability to disrupt tauopathy and also improved traumatic brain injury (TBI) – induced cognitive/memory deficits in vivo (Figure 5). The results overall demonstrated that the modified circular aptamer can be further developed into diagnostic and therapeutic candidates for tauopathies.

Figure 5. Top images – Ex vivo fluorescence of mice injected with Cy5.5 labeled circular aptamer, Tau aptamer and TfR aptamer for different time periods. Bottom images – Therapeutic effect of circular aptamer on tauopathy treatment evaluated by ELISA assay post controlled corticol impact (CCI) surgery.

In conclusion, aptamers have demonstrated the clinical need for targeted treatment of brain and neurological diseases. With the propensity to cross the BBB, aptamers have also shown to target specific cell or signaling systems, respond to endogenous stimuli, or act as vehicles for gene delivery. The ability to easily modify aptamers by attaching various functional groups allows creation of aptamer complexes with different drugs and nanomaterials, enhancing their functions.

Aptamer Group Ltd (AGL) offers the advantage of designing aptamers to be optimized for the conditions you want to use them in. This way they are engineered to bind to their target with high specificity and affinity. AGL has dedicated researchers to identify aptamers against novel and significant targets and continuously aims to conduct further research in prevention, diagnosis and treatment of cancer and neurodegenerative diseases. If you would like to know more about aptamers and their applications in diagnostics and therapeutics, please contact us using the form below.

References:

Bukari B, Samarasinghe RM, Noibanchong J, Shigdar SL. Non-Invasive Delivery of Therapeutics into the Brain: The Potential of Aptamers for Targeted Delivery. Biomedicines. 2020;8(5):120. Published 2020 May 14. doi:10.3390/biomedicines8050120

Li X, Yang Y, Zhao H, et al. Enhanced in Vivo Blood-Brain Barrier Penetration by Circular Tau-Transferrin Receptor Bifunctional Aptamer for Tauopathy Therapy. J Am Chem Soc. 2020;142(8):3862-3872. doi:10.1021/jacs.9b11490

Shi S, Fu W, Lin S, et al. Targeted and effective glioblastoma therapy via aptamer-modified tetrahedral framework nucleic acid-paclitaxel nanoconjugates that can pass the blood brain barrier. Nanomedicine. 2019;21:102061. doi:10.1016/j.nano.2019.102061

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