Precision Engineering nanoparticles for drug delivery

“This is where formulations come to die!” These are words of a prominent pharmaceutical company GMP formulations lead. What makes this so difficult? How can a formulation go this far before falling over? Presumably there have been extensive clinical trials, yet as soon as scaling is called for something fails. What makes a winning formulation? This article will explore some key points to consider when formulating medicinal products, predominantly for in vivo injection. 

Many years ago, at a conference in Chicago, a pharmaceutical company representative asked advice on how to analyse a tablet post digestion – because they ‘had no idea what really happens to their oral medications once swallowed’. This sent me into a spin… Since then, I have sought alternatives for drug delivery.

Intravenous (IV), subcutaneous (SC), and Intramuscular (IM) routes help reduce the actives’ concentration requirement plus preserves the payload from the oral degradation. They enjoy a rapid onset of action, are apparently a predictable way of action and almost complete bioavailability. Given the nature of the payloads considered, this chapter will focus on routes other than oral.

How do we encapsulate nanomedicines for targeted delivery

Common reasons to encapsulate Protein, peptide, RNA, or small molecules are many, such as to protect the payload and to facilitate cellular uptake. Other proposed uses include the incorporation of a ‘decoration’ to guide the nanoparticle to a particular target, though there is little evidence of this being very specific. Novel nanoparticles looking at changes in structure and constituents are increasingly being employed. Importantly a nanocarrier will reduce systemic toxicity, reduce concentrations of the payload required, and protect a payload from premature biological degradation. 

How can we currently encapsulate?

Over the years there has been a dramatic increase in microfluidic devices from an array of companies attempting to cash in on the explosion of research, particularly with RNA of one flavour or another.
I think it was Harry Truman who stated, “If you can’t convince them, confuse them”, I aim to demystify this expanding world and hopefully bring clarity to this confusion.  If you invest time in reading the white paper https://www.atascientific.com.au/a-better-way-to-create-nanoparticles/ you will find a discussion about an array of microfluidic devices, how they work and the limit of their uses. To expand, this document relates to the formulation itself and its interaction with mixer employed. 

Formulation as a function of prevailing technology

Many companies and research institutes invest a deal of resources formulating a lipid construct that will not only encapsulate the required cargo but mix well to elicit the targeted size and Poly Dispersity Index. This is the current paradigm. Unfortunately, each mixer has a peculiar method where, at times, the formulation fails with orthogonal technologies or fails to scale efficiently. Some organisations invest in low-cost T Mixers or similar only to learn that they need to spend enormous human resources to develop a formulation to efficiently produce poultry volumes. In the Australian parlance, this is an apt descriptor for “the tail wagging the dog”. Ideally the most versatile technology would be one that has the capacity to morph to a formulation. 

If I had a winning formulation, I would be reticent to modify it for a mixer. I recall a conversation I had with the Director of CMC in a Canadian therapeutics’ organisation, he stated “We make exceptional formulations; You just have to mix it”. Whilst it sounds flippant, he was prompt to add, the technology we presented was adept at transforming to suit any given formulation, indeed in “two runs” we had nailed a formulation they had to tweak for 6 months on another technology.

It is becoming increasingly clear that formulations may be stymied by the technology employed. The inflexibility of many microfluidic devices coupled with the abject failure to scale is concerning. The lure of an easy fix or popular choice of peers may in fact limit the very research you wish to expand, not only in terms of expense, time, and resource but opportunity. Many formulations are likely to have failed as they approached the limit capacity of the employed mixer.  Following the golden rules of nanoparticle creation- mixing rate must be faster than assembly rate.  

Nanoprecipitation can be tuned with changes to flow rate and the flow rate ratio of the two phases mixed. Well, this is the case with most instruments, what if you can make minor modifications of the technology to enhance the creation of bespoke formulations, potentially turning what was thought to be marginal into a lead candidate. In the discussion below about PEG alternatives, I pondered their acceptable results and wondered what if these were a function of the technology limit- not the lipid’s limit? Frequently I have run formulations where microfluidics yield Encapsulation Efficiency (EE) of around 80– 90% – acceptable? Not really as I approach 99 – 100% on the exact same formulation. 

Quality of nanoparticles

Assessing the apparent quality of a nanoparticle is interesting! To what measure or standard?
The current theory starts with Dynamic Light Scattering with the gold standard system – the Malvern Panalytical Zetasizer, analysing the Size, Poly Dispersity Index (PDI) and ZetaPotential. The next question is the Encapsulation Efficiency (EE) a measure of how much of the payload eg API or RNA is actually held within the construct of the nanoparticle. Ideally it is as close to 100% as possible as the cost of some payloads are significant, where every 1% reduction in EE will have dramatic effects on total costs of production. – if you are not encapsulating you are virtually flushing away the expensive RNA. There needs to be more research into analytics of the payload particularly what is the effect on the payload once it is encapsulated into a nanoparticle? Such needs are heightened should multiple APIs be encapsulated within the same nanoparticle, what interactions occur? Some interesting research into this arena with RNA is occurring with the advent of Microfluidic Modulation Spectroscopy, a technology capable of elucidating protein structure. Recently an enhancement enabled easy detection and comparison of RNA changes that are vital for therapeutic development, and here is the kicker – within an LNP.

Checking the in-vivo capabilities of the particle is where ‘the rubber hits the road’. Understanding transfection, bio-distribution and efficacy is the next level of assessment. Clearly this will define the effectiveness of the delivery vehicle and the payload as well. A long-held debate has been whether the murine model is a valid measure. A recent paper from the University of British Columbia (UBC) has some interesting findings with a porcine model¹, demonstrating “… that following a low-dose infusion of mRNA-LNP, exogenous protein and exogenous mRNA transcripts can be detected globally including in the liver, spleen, lung, heart, uterus, colon, stomach, kidney, small intestine, and brain of the swine. Notably, we also detected exogenous protein expression in the bone marrow, including megakaryocytes, hematopoietic stem cells (HSC), granulocytes, and in circulating blood cells such as white blood cells and platelets.”¹ It has, in the past, been widely accepted to attain extrahepatic targeting employment of a specialised moiety decorating the surface of a Lipid NanoParticle (LNP) is required, perhaps this may not be a necessity. 

Not all Lipids are equal

The complexity of formulation does not end with the achievement of appropriate particle size, PDI, zetapotential and EE. Even with an EE of 99% the question remains whether there is adequate cellular uptake, does the payload release where intended, is it taken up by the intracellular organelles targeted, what happens in endosomal escape? Most of these are poorly understood. Screening of a lipid candidate can include biodistribution studies where expression of a fluorescent marker is tracked by imaging, or alternate methods. Perhaps it is the formation of antibodies that define success. “Despite the developments in the LNP field, a commonly overlooked aspect regards the very limited release of the nucleic acid payloads in the cytoplasm¹⁰.”

“The highly dynamic nature of endosomal compartments and shared markers between them adds another layer of complexity making it even more difficult to gain common conclusion regarding trafficking and escape of LNPs. Nevertheless, endosomal escape is one of the most important aspects for efficient nucleic acid delivery, and it is also pertinent to pay attention to the requirement of sophisticated tools and probes to understand the basic molecular biology of this process¹⁰.”  

Lipids may not be equal, neither are the payloads and their purpose. It has been noted that there are many lipids that combine to form an LNP, as Robin Shattock’s group¹¹ reports, the helper lipid selection can have a significant role.  They noted ‘helper lipid identity altered saRNA expression’ and ‘LNP storage’. Confirming formulation is a richly diverse field with a great deal to contribute to medicine. 

It may not be just about vaccination or chasing a tumour, it may be as fundamental as enabling better platelet transfusions. Exciting research at UBC has succeeded in transfecting platelets with mRNA via an LNP, to ‘enable exogenous protein expression in human and rat platelets’¹². It is thought to be the first instance of this and could possibly expand the therapeutic potential of platelets. 

Alternate Formulations

Metal-phenolic networks (MPN) are offering another indication there could be alternatives to the current paradigm. The Caruso lab in Melbourne University have developed a method to create MPN Nanoparticles² in not only a straightforward reproducible, but tunable manner. “We demonstrate the role of buffers (e.g., phosphate buffer) in governing NP formation and the engineering of the NP physicochemical properties (e.g., tunable sizes from 50 to 270 nm) by altering the assembly conditions. A library of MPN NPs is prepared using natural polyphenols and various metal ions. Diverse functional cargos, including anticancer drugs and proteins with different molecular weights and isoelectric points, are readily loaded within the NPs for various applications (e.g., biocatalysis, therapeutic delivery) by direct mixing, without surface modification, owing to the strong affinity of polyphenols to various guest molecules.²” This is hugely impactful work with global implications. Well worth following this lab for the exciting things to come. 

Polysarcosine lipids – PEG alternative

Lipid nanoparticles (LNPs) such as ALC-0315 and SM-102 LNPs have been applied to deliver mRNAs encoding viral antigens against the SARS-CoV-2 virus. These LNPs are typically comprised of four distinct lipid components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol lipids (PEG lipids)³. Prior to the COVID Pandemic there were studies evaluating the prevalence of pre-existing anti-PEG antibodies⁴. The use of this lipid construct across the globe to vaccinate billions of people prompted over a dozen studies regarding anti-PEG antibodies occurrence, defining which immunoglobulin was triggered and resultant allergic responses. As reported by Kang³ the results were conflicting. Despite the inconclusive nature of these studies, there is a growing appeal for alternatives to PEG.  Kang³ evaluated a panel of polysarcosine (pSar) as a direct replacement to PEG in the ALC-0315 and SM-102 based LNP formulations with comparable physicochemical properties perhaps even enhancing mRNA delivery efficiency in-vitro and in-vivo along with overall immunogenicity. Interesting developments that may assist all LNPs. 

Poly-lactic-co-glycolic acid (PLGA)

PLGA are a family of FDA-approved biodegradable polymers that are physically strong and highly biocompatible and have been extensively studied as delivery vehicles of drugs, proteins, and macromolecules such as DNA and RNA⁶. Over the years there have been few more enduring materials in drug delivery research with such a wide range of applicability than that of Poly-lactic-co-glycolic acid (PLGA). Such a wonderful biodegradable polymer that essentially breaks down to Lactic and Galactic acids and ultimately CO₂ and H₂O eliminated by the human body through a natural pathway such as the Krebs cycle. As diverse as the applications, so are the methods to create PLGA particles, such as Spray drying, Microfluidics and nanoprecipitation with lipids to name just a few. Most methods struggle to create enviable polydispersity and volumetric yield. The methods are laborious and difficult to scale. A solution to this is the Advanced Cross Flow (AXF)⁵ where Membrane emulsification operates in a controlled low shear environment, bringing the advantages of microfluidic mixing to the commercial scale. By substantially reducing the energy input membrane emulsification dramatically improves particle size distribution when compared to traditional batch homogenization. The result is a more consistent product that is not lost to downstream filtration. 

Moreover, AXF technology operates continuously, enabling real time process monitoring and feedback control during drug product manufacturing. A simple change to the membrane tunes the system to produce either nano or Micron sized particles. One installed system has been configured to create 10 Kg of PLGA particles per day, dispelling the fear of lack of yield. In a search Aug 2024, of ClinicalTrials.gov (https://clinicaltrials.gov/search?term=PLGA&intr=PLGA ) there were 43 with PLGA particles.

Freeze dried lyophilised 

Whilst not strictly a novel formulation method, freeze drying is interesting as a delivery vehicle for other formulations. Powdered forms of nanoparticles could be highly efficient nasal delivery systems.
“The lung is directly exposed to the outside environment through the airways. It contains two main functional parts, the conducting zone (trachea, bronchi and bronchioles) and respiratory zone (alveoli). The top five most common lung diseases causing severe illness and death worldwide include tuberculosis, respiratory infections, lung cancer, asthma and chronic obstructive pulmonary disease (COPD), which together have a huge global burden⁷.” Imagine the global health significance of a loaded LNP, freeze-dried and delivered by inhaler direct to the lungs. Sending anything into the lungs is not as simple as it sounds. Consider that at its core, the lung is a site for gaseous exchange, but unfettered access would expose the body to innumerable airborne pathogens.

 Evolution has armed us with some formidable defenses such as a wash of antimicrobials, mucus, neutralising immunoglobulins, beating cilia and an epithelial cell layer. These are bolstered by white blood cells. This is why packaging the drug to move by stealth enhances the success of treatment.

Liquid Crystalline Nanoparticles (LCNP)

Distinctive structural features of lyotropic nonlamellar liquid crystalline nanoparticles (LCNPs), such as Cubosomes and Hexosomes enhance their applicability as effective drug delivery systems. “Cubosomes have a lipid bilayer that makes a membrane lattice with two water channels that are intertwined. Hexosomes are inverse hexagonal phases made of an infinite number of hexagonal lattices that are tightly connected with water channels. These nanostructures are often stabilized by surfactants. The structure’s membrane has a much larger surface area than that of other lipid nanoparticles, which makes it possible to load therapeutic molecules. In addition, the composition of mesophases can be modified by pore diameters, thus influencing drug release⁸”. This method has the potential for a kind of repackaging of existing medications for more efficient and effective delivery.
In context- antimicrobial drugs may be able to pass into cells, therefore the previously unreachable intracellular bacteria can be treated. Consider chronic lung infections. An example of how effective this is was the subject of a study at the University of South Australia, ´The ability of a cationic LCNP to improve the performance of antibiotics against intracellular bacteria present in macrophage and epithelial cells was determined. The presence of DDAB altered the morphology of LCNPs into an onion-ring like structure. The inclusion of a cationic lipid enhanced cellular uptake, >50% and > 90% in macrophages and epithelial cells, respectively. In macrophages, LCNP-DDAB reduced intracellular P. aeruginosa and S. aureus viability by ∼ 90% and ∼ 55% at 4x MIC of antibiotics.⁹’

Looking toward the future of nanoparticle formulation

Will there be one magic bullet or horses for courses approach? If history boasts a rich tapestry of methods for drug delivery, the future is sure to be exciting. This review is not definitive, it is moreover a snapshot of a few prevalent procedures in a constantly evolving environment.

The creation of nanoparticles has nuances, being able to pivot to these with a flexible technology will likely be the successful combination going forward. Microfluidic devices have challenges when called upon to scale up despite the promise of a limited interventionary technique. Their failure has prompted a paradigm shift to a novel method of nanoparticle production, the Micropore Technologies Advanced Cross Flow systems are a solution with ample flexibility to take you from 200 ul R&D samples to thousands of litres in GMP production using the same technology in R&D as in GMP without being formulation centric.  Whilst not a panacea for all delivery goals, this technology sure has legs for the lions’ share.

References

1. ´Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine’ Ferraresso et al 2024 Molecular Therapy: Methods & Clinical Development https://doi.org/10.1016/j.omtm.2024.101314
2. ‘Direct Assembly of Metal-Phenolic Network Nanoparticles for Biomedical Applications’ Xu et al. Angewandte Chemie International Edition https://doi.org/10.1002/anie.202312925
3. ‘Engineering LNPs with polysarcosine lipids for mRNA delivery’ Kang Et Al, Bioactive Materials 37 (2024) 86-93. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10957522/pdf/main.pdf
4. Analysis of Pre-existing IgG and IgM Antibodies against Polyethylene Glycol (PEG) in the General Population Yang . Q. Et Al Anal Chem. 2016 Dec 6;88(23):11804-11812.
   doi: 10.1021/acs.analchem.6b03437. Epub 2016 Nov 16.
5. ‘Polymeric Encapsulation of Active Pharmaceutical Ingredients’ https://microporetech.com/applications/biodegradable-polymeric-materials
6. ‘PLGA-Based Nanomedicine: History of Advancement and Development in Clinical Applications of Multiple Diseases’ Alsaabet Al, Pharmaceutics. 2022 Dec; 14(12): 2728 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9786338/
7. ‘Lipid Nanoparticles as Delivery Vehicles for Inhaled Therapeutics’ Leong et al Biomedicines. 2022 Sep; 10(9): 2179. doi: 10.3390/biomedicines10092179
8. ‘Recent Advances in the Development of Liquid Crystalline Nanoparticles as Drug Delivery Systems’ Leu et al Pharmaceutics. 2023 May; 15(5): 1421.
   doi: 10.3390/pharmaceutics15051421
9. ‘Liquid crystalline lipid nanoparticles improve the antibacterial activity of tobramycin and vancomycin against intracellular Pseudomonas aeruginosa and Staphylococcus aureus’ Subramaniam et al International Journal of Pharmaceutics Volume 639, 25 May 2023, 122927, https://www.sciencedirect.com/science/article/pii/S0378517323003472#b0240
10.  ‘Endosomal escape: A bottleneck for LNP-mediated therapeutics’ Chatterjee et al; PNAS March 2024: https://doi.org/10.1073/pnas.2307800120
11. ‘The role of helper lipids in optimising nanoparticle formulations of self-amplifying RNA´ Barbieri et al; Journal of Controlled Release Volume 374 , October 2024, Pages 280-292 https://doi.org/10.1016/j.jconrel.2024.08.016
12. ‘Genetically engineered transfusable platelets using mRNA lipid nanoparticles’ Leung et al;
    Sci. Adv. 9, eadi0508 (2023), Downloaded from https://www.science.org on September 02, 2024