Capsosomes: the revolutionary enzyme carriers

In the case of any disease, having access to the right drug can make all the difference and having the adequate drug is one of the most important steps in disease treatment. However, merely having the drug is worthless if it never reaches the site of action. For this reason, equipping pharmaceuticals with a vehicle to make this possible is important; which is why there is a whole field of research around drug delivery. Designing an appropriate drug carrier is not an easy task. The body environment is, in general, very hostile to any substance that is not natural to the organism – the so-called xenobiotics – or that comes from an unusual route of administration. The digestive tract, for example, is an especially tough environment, with different pH areas and lots of proteases lurking in it. These levels of protection are generally a positive feature and one of the reasons we make it through to old age. However, with our body being such an efficient sentinel, it is hard for many drugs to reach its site of action unscathed.

That’s not to say that overcoming those barriers is entirely difficult; it depends on the case. For drugs that are meant to act quickly and punctually, like anti-inflammatories, it is sufficient to protect the active ingredient, so it reaches its site of action. Once there, it gets degraded and, after approximately six hours, the substance is presumably disintegrated in full: its job is done, and the drug is gone. However, in other cases it is more complicated than that. Lysosomal storage disease, which is characterised by the body’s inability to produce certain enzymes, is one such example. For sufferers, the lack of a particular enzyme is critical and leads to severe problems, as their body is unable to perform certain essential physiological activities. In many cases, life expectancy is extremely low. For those affected, provision of the missing enzymes solves the problem for a short period, but a single administration isn’t sufficient. As the saying goes, “give a man a fish and you feed him for a day, teach a man to fish and you feed him for a lifetime”. In this case, teaching the man to fish would be the equivalent of providing the patient’s body with the possibility of generating its own enzymes. While this presents an interesting challenge for scientists in the field of gene therapy, there is still a long way to go until a proper solution is achieved.

This begs the question, how do people with enzyme deficiencies deal with their diseases? In the case of phenylketonuria – a disease characterised by the lack of the enzyme that breaks phenylalanine into tyrosine – people normally opt for a phenylalanine-free diet to avoid complications. However, a research study showed that half of these patients struggle to stick to a controlled diet,¹ and in any case it is not an optimal solution. For other lysosomal storage diseases like Fabry disease or Niemman-Pick disease type C, patients must resort to enzyme replacement therapies, which imply the periodic inoculation of the missing enzymes.^2,3 The lack of a proper biocompatible drug carrier that protects enzymes through to their site of action, impairs the lives of those who suffer from these diseases.particular enzyme is critical and leads to severe problems, as their body is unable to perform certain essential physiological activities. In many cases, life expectancy is extremely low. For those affected, provision of the missing enzymes solves the problem for a short period, but a single administration isn’t sufficient. As the saying goes, “give a man a fish and you feed him for a day, teach a man to fish and you feed him for a lifetime”. In this case, teaching the man to fish would be the equivalent of providing the patient’s body with the possibility of generating its own enzymes. While this presents an interesting challenge for scientists in the field of gene therapy, there is still a long way to go until a proper solution is achieved.

Light at the end of the tunnel

In order to tackle this problem, scientists were inspired by nature. They asked themselves where enzymes are naturally stored in the body and noted that cells possess small sacs called lysosomes to retain their structural integrity, as enzymes don’t cope alone in the cytoplasm. From this observation, scientists decided to try designing artificial lysosomes to carry the missing enzymes into the organism. Liposomes have proven to be good structures to encapsulate enzymes while retaining their activity,⁴ but to provide a sufficient number of enzymes to replace the activity of the missing ones would require several liposomes. This prompted the idea for capsosomes.

Capsosomes are polymeric carrier capsules that contain liposomes as sub-cargo structures.⁵ Although there are several types of multi-compartmentalised vesicles, the combination of hydrophilic and hydrophobic systems affords capsosomes the characteristics of both types. While liposomes protect the enzymatic structure from misfolding or denaturation, small substrates can enter and leave the structure through the lipid bilayer allowing the enzyme to perform its activity over its target substrate.⁴ Meanwhile, the polymeric structure, while also semipermeable, confers structural protection to the liposomes, which are more susceptible to degradation in this environment.

Capsosomes are synthesised using the layer-by-layer technique. This involves the deposition of alternate layers of differently charged polyelectrolytes in order to build each layer around a silica core, to then attach the liposomes.⁶ The silica core can be removed after the structure is built. Using this technique, capsosomes can range in size from nanometres to a few micrometres. For instance, capsosomes with a diameter of 3µm can contain more than 150,000 liposomes between their polymeric layers.⁷ Due to their structure, enzymatic substrates can permeate through liposomes at body temperature.⁸ In this way, capsosomes not only act as enzymatic vehicles, but as micro-reactors inside the body.

While its clinical efficacy is yet to be proved, in vitro experiments are showing promising results. In 2015, a group of researchers from the University of Aarhus in Denmark and the National University of Singapore successfully assembled enzyme-loaded capsosomes in order to degrade alanine into trans-cinnamic acid.⁸ The group, led by Letícia Hosta-Rigau and Brigitte Städler, demonstrated that capsosomes can co-encapsulate different enzymes and allow consecutive reactions to occur inside. Also in 2015, a team from the University of Melbourne in Australia, led by Frank Caruso and James W. Maina, demonstrated the ability of capsosomes to perform protein loading and release in physiological conditions for more than two months, portraying capsosomes as potential candidates for sustained release.⁹

If future clinical trials show positive results, capsosomes could revolutionise the medical field. The synergy between synthetic chemistry and molecular biology could provide insights into enzyme-replacement therapies, showcasing the importance of drug formulation in such treatments.

One of the main reasons for success is that capsosomes were inspired by natural structures. The creation of multi-compartmentalised structures is an example of cell mimicry – a field dedicated to the design of artificial cell structures and, ultimately, artificial cells. This is a promising scenario that demonstrates how replacement of defective or missing cell structures could be solved with the design of artificial but nature-inspired cellular structures. Coming back to the earlier analogy, for a man who can’t learn to fish, capsosomes at least ensure that fish reaches his belly.

Published at European Pharmaceutical Review (July 2019). Link: https://www.europeanpharmaceuticalreview.com/article/91096/capsosomes-the-revolutionary-enzyme-carriers/

References

1. Gassió R, Campistol J, Vilaseca M, Lambruschini N, Cambra F, Fusté E. Do adult patients with phenylketonuria improve their quality of life after introduction/resumption of a phenylalanine-restricted diet? Acta Paediatrica 2007;92:1474–8.
2. Fabry disease – Genetics Home Reference – NIH. US National Library of Medicine. https://ghr.nlm.nih.gov/condition/fabry-disease (accessed May 25, 2019).
3. Sedel F. Niemann-Pick Disease Type C. Oxford Medicine Online 2016. doi:10.1093/med/9780199972135.003.0053.
4. Jahadi M, Khosravi-Darani K. Liposomal Encapsulation Enzymes: From Medical Applications to Kinetic Characteristics. Mini-Reviews in Medicinal Chemistry 2017;17:366–70.
5. Teo BM, Hosta-Rigau L, Lynge ME, Städler B. Liposome-containing polymer films and colloidal assemblies towards biomedical applications. Nanoscale 2014;6:6426.
6. Picart C, Caruso F, Voegel J-C. Layer-by-layer films for biomedical applications. Weinheim, Germany: Wiley-VCH Verlag; 2015.
7. Yoo CY, Seong JS, Park SN. Preparation of novel capsosome with liposomal core by layer-by-Layer self-assembly of sodium hyaluronate and chitosan. Colloids and Surfaces B: Biointerfaces 2016;144:99–107. doi:10.1016/j.colsurfb.2016.04.010.
8. Hosta-Rigau L, York-Duran MJ, Kang TS, Städler B. Extracellular Microreactor for the Depletion of Phenylalanine Toward Phenylketonuria Treatment. Advanced Functional Materials 2015;25:3860–9. doi:10.1002/adfm.201404180.
9. Maina JW, Richardson JJ, Chandrawati R, Kempe K, Koeverden MPV, Caruso F. Capsosomes as Long-Term Delivery Vehicles for Protein Therapeutics. Langmuir 2015;31:7776–81.

B. cepacia: What is it and Why is it a Concern?

Hospitals and Pharmaceutical Plants on the Prowl for this Uninvited Guest

Burkholderia cepacia has been in the news...again!

Even though this pathogen is not a newcomer (one can easily findpapers from the 1990s discussing its dangers), it continues to wreak havoc in pharmaceutical plants (1). Less than two years ago, two separate batches of oral solid medicine were found to be contaminated by B. cepacia (2,3). The year before,a manufacturer issued a Class I recall for saline flush IV syringes suspected to be responsible for B. cepacia bloodstream infections (4). A quick Web search shows these are not isolated cases. B. cepacia is a major contaminant in both sterile and nonsterile products. A review of U.S. FDA recall data from January 2012 to July 2012 found that 39% of contamination cases in non sterile products were due to the presence of B. cepacia (5).

Before continuing, one might wonder, what is B. cepacia? B. cepacia is actually the name given to a group of Gram negative aerobic rod-shaped bacteria (Burkholderia Cepacia Complex or BCC). Despite living mainly in aerobic environments, those bacteria can survive in hypoxic conditions and possess the ability to grow in low nutrient media as well. This microorganism is generally harmless to healthy individuals but acts as an opportunistic pathogen, presenting a potentially fatal danger to immunocompromised patients. A recent study performed in Severance Hospital at Yosei University College of Medicine in Korea found a mortality rate of 41% among individuals suffering of B. cepacia-induced bacteremia (6). This is a problematic issue as this pathogen is quite proficient at reaching sick individuals through two main channels: hospital fluids and contaminated pharmaceutical products (7).

A Hardy, Vicious Microorganism

There is another feature of B. cepacia worth to highlight, and this is its proficiency at developing a biofilm matrix. Biofilm forming organisms feature a wide range of bacteria, including mainly Gram negative but also Gram positive pathogens (8,9). These microorganisms are generally quite hardy since the biofilm acts as a net that stops antibiotics from penetrating into the actual bacterial cell, exposing only the planktonic cells outside the matrix. (10). For this reason, bacteria communities that form biofilms show a higher than average resistance to antibiotics, stress and many other conditions that are usually detrimental to cells (11). All in all, it makes dealing with biofilm-forming bacteria difficult, especially those inside an individual’s body.

A famous example of a biofilm-related disease would be cystic fibrosis, which is mainly caused by Pseudomonas aeruginosa—a bacterium with a long, well-known association with B. cepacia (in fact, B. cepacia was initially known as Pseudomonas cepacia) (12). Even though changes in antibiotic administration patterns (like alternate antibiotic cycles or combined antibiotics administration) can significantly reduce its mortality, many biofilm infections are considered chronic, as a complete elimination of the pathogen from the host is very difficult. This, coupled with the great difficulty in detecting it via conventional methods, makes it a really unpleasant microorganism to deal with. Naturally,limiting its chances to reach hospitals is vital to avoid a problem that presents huge human and economic costs.

Manufacturers Fight B. cepacia

But it is not only hospitals that are involved in this crusade against B. cepacia. Pharmaceutical companies have also a huge interest in eradicating this pathogen from their facilities, as its very presence forces production to stop, risking potential loss of clients, and even causing severe economic losses in some cases. As mentioned before, fighting a biofilm-inducing bacterium inside a human host can be a complicated task. Yet eradicating its presence in a plant’s water systems might not be as difficult. Fortunately, the industrial environment allows for use of more aggressive methods and chemicals. Some new products have been appearing on the market, offering potential solutions that are more efficient solutions than conventional bactericides. Many scientific teams are focusing on new approaches to eradicate B.cepacia (13). Still, present solutions have, so far, failed at eliminating B. cepacia completely from both infected humans and industrial water conducts.

Two-Front Approach to B. cepacia

Risk management of B. cepacia contamination can be tackled from two different perspectives. There is the hospital approach, which focuses on good hygiene and patient segregation based on their microbiological status, and there is the industrial approach, which is based on detecting this microorganism efficiently enough to avoid contaminated products reaching the market.

Detecting B. cepacia presence is a hard task. B. cepacia is a bacterium that usually presents slow growth and many times remains undetected until it is too late (14,15). USP considered this matter critical enough as to dedicate a general chapter—which is currently undergoing an In-Process Revision—featuring tests for growth promoting, indicative and inhibitory properties of the media and a list of recommended media for the tests, and clues for the interpretation of possible B. cepacia colonies (16). In addition, some newer techniques like real-time polymerase chain reaction are being used in order to speed up the detection process (17). It is also true, however, that these techniques, while being quicker than the traditional methods, are still relatively time consuming compared to others.

The development of an in situ media-independent device to detect B. cepacia in water (something similar to point-of-care devices in hospitals) could mean a big step toward better, quicker detection. Meanwhile, detection remains problematic. In order to lower the risks of B. cepacia spreading to the general public, it is recommendable to invest money on efficient detection systems for B. cepacia early detection. It is also highly advisable to thoroughly disinfect, especially in cases where B. cepacia or other biofilm-forming bacteria have been spotted.

Knowing the gravity of the situation, it might seem logical to think that B. cepacia should be considered a serious threat and be dealt with properly. In the end, tackling the problem at its source will save many lives and millions for both pharmaceutical plants and hospitals.

Published at PDA Letter (April 2019). Link: https://pda.inloop.com/en/article/114233/b-cepacia-what-is-it-and-why-is-it-a-concern

References

1. Koenig, D. W., Mishra, S. K., and Pierson, D.L. “Removal of Burkholderia cepacia biofilm swith oxidants.” Biofouling 9 (1995): 51-62.
2. Office of Regulatory Affairs. (2017, August 2). Recalls, Market Withdrawals, & Safety Alerts Rugby Laboratories Issues Voluntary Nation-wide Recall of Diocto Liquid and Diocto Syrup Manufactured By PharmaTech, LLC Due to Possible Product Contamination
3. Office of Regulatory Affairs. (2017, August 30). Recalls, Market Withdrawals, & Safety Alerts - Mid Valley Pharmaceutical LLC Issues Voluntary Recall of Doctor Manzanilla Cough& Cold and Doctor Manzanilla Allergy &Decongestant Relief Syrup Due to Potential Contamination with Burkholderia Cepacia.
4. Center for Devices and Radiological Health. (2016, October 4). Medical Device Recalls Nurse Assist Inc. Recalls Normal Saline Flush IV Syringes Due to Possible Burkholderia Cepacia Bloodstream Infections.
5. Ali, M. “Burkholderia Cepacia in Pharmaceutical Industries.” International Journal of Vaccines & Vaccination, 3 (2016)
6. Ku, N. S., et al. “Risk factors for mortality inpatients with Burkholderia cepacia complex bacteraemia.” Scandinavian Journal of Infectious Diseases, 43 (2011): 792-797.
7. Mahenthiralingam, E., et al. “The multifarious, multireplicon Burkholderia cepacia complex.” Nature Reviews Microbiology 3 (2005): 144-156.
8. Schembri, M. A., et al. “An Attractive Surface:Gram-Negative Bacterial Biofilms.” Science Signaling (2002) 132.
9. Abee, T., et al. “Biofilm formation and dispersal in Gram-positive bacteria.” Current Opinion in Biotechnology 22 (2011): 172-179
10. Murphy, M. P., and Caraher, E. “Residence in biofilms allows Burkholderia cepacia complex(Bcc) bacteria to evade the anti-microbial activities of neutrophil-like dHL60 cells.” Pathogens and Disease (2015).
11. Gough, N. R. “Complexity in the Bacterial Community.” Science Signaling (2008).
12. “Identification of the Burkholderia cepacia complex by PCR from isolated cultures or direct analysis of sputum from cystic fibrosis patients in Brazil.” Journal of Cystic Fibrosis (2005): 4
13. Sousa, S. A., et al. “Post-genomic Approaches and Bioinformatics Tools to Advance the Development of Vaccines against Bacteria of the Burkholderia cepacia Complex.” Vaccines 6 (2018).
14. Pope, C. F., et al. “Approaches to measure the fitness of Burkholderia cepacia complex isolates.” Journal of Medical Microbiology, 59 (2010): 679-686.
15. Govan, J. R., et al. “Common Questions About Burkholderia cepacia” UGent.
16. <60> MICROBIOLOGICAL EXAMINA-TION OF NONSTERILE PRODUCTS—TESTS FOR BURKHOLDERIA CEPACIACOMPLEX.” USP Pharmacopeial Forum, Sept. 2018,
17. Lowe, C., et al., “A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis.” Plos One 11 (2016).