Building a Microscopy facility must be a daunting task, with such an array of imaging technologies and applications. Gone are the days of the humble compound microscope whose predecessor illuminated the ‘cell’ as coined by Robert Hooke¹. I can only imagine the excitement of such a discovery and ponder how many times microscopy has yielded such emotion since then. Microscopy unlocks a window into a wondrous world, there is no debate around its value, just what is the most useful. 

It is overly simplistic to consider that there need only a few technologies to fulfil the gambit of application needs. As with most things, it’s a function of ‘horses for courses’ as one technology is great at specific functions though not so fantastic at others. As technology marches on, the shiny new ‘must have’ attracts the researcher desperate to publish novel findings elucidated by a magical black box developed by a clever physicist. This hunger is fed by the perennial ‘publish or perish’ mantra exacerbated by the anecdotal tendency for novel technology to gain favourable acceptance by journals. The issue of competing interests should not be overlooked.

The Arms Race

The arms race in microscopy is not just driven by the researcher. Consider the microscope manufacturers desire, a FOMO, adding their flair to a particular development they have not invented but want to have a me-too with added ‘bells and whistles’ to gain the upper hand in a sale and capitalising on brand loyalties. Super-resolution microscopy is a great example of this, by delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques have opened new opportunities to study biological structures with details approaching molecular structure sizes² – note STED, SSIM , PALM, STORM and RESOLFT. Bending the frontier delving beyond what was thought impossible. Beautiful discoveries, however, it still has limitations in capabilities. 

A divergence is occurring, a whole new world of computational microscopy is emerging to enhance the image we no longer see. This concept is spawning a plethora of variations on the theme in the race to the ultimate in resolution. Whilst many are all-consumed by this resolution race, others are inspired by not just the individual, but the population – the company they keep in equal measure. We can all plead guilty to only seeing the majority and overlook the outlier – visualise a cluster in a flow cytometry experiment and consider the gating applied to the data along with the premise of these actions. This may well be the cell that is going to kill the patient. 

Why bother?

Perhaps it is time we consider a better way of investigating the behaviour of live cells – a novel method of looking at the cells in question that has a very large field of view to enhance the statistics, a way to view them for extended time frames, even weeks, without perturbing, killing, or bleaching. A gentle nourishing environment to keep cells happy and label-free is ideal. You may be thinking – Why bother given there are so many options for microscopes? In short, signal to noise ratio is limited when using standard microscopy methods. While the ubiquitous use of fluorescence may seem to have solved this, new data is emerging that, arguably and at times unknowingly, has exposed fluorescence as a major influencer of cell behaviours itself. Rarely can you gain such powerful insight with incredible single cell tracking and metrics right through to entire populations using Fluorescence alone. 

How does Ptychography work? 

Ptychography is not only an unusual name, but an unusual technology. It is a hybrid of sorts, a blend between an optical microscope and a light scattering detector. Briefly, the optical microscope sends light through a sample and a scatter or diffraction pattern is received on the detector, a digital method is used to translate the diffraction pattern into a quantitative image this is the Phasefocus virtual lens. The patterns are processed by the Ptychography algorithm to produce quantitative intensity and phase images of the sample. To explore this further please click here³.

The resultant is a dramatic increase in the Signal: Noise giving the opportunity to avoid fluorescent probes, enabling a very low intensity light source avoiding cellular damage. This is a big win for the cells and the researcher trying to see them in an environment as natural as possible.

A Bitter Pill

It is tough to acknowledge that the core method of Fluorescence employed by so many in cell biology may be delivering flawed results. Several studies run on cells in parallel with and without a fluorescent label have shown profound changes to behaviours and other indicators such as proliferation, motility, and dry mass, prompting the question – Why aren’t more facilities using this technology if only for a confirmatory application notwithstanding the enormity of insight it offers beyond this? One could postulate it may be because users are blinded by resolution.

What am I likely to see in a facility?

Summarising the breadth of microscopes with their strengths and weaknesses is not a simple task and in doing so assumptions must be made along with the lens we look through. The image below gives a summary, but more importantly, it shows where Livecyte fits into the scheme of a broader microscopy facility. It is worth noting, Livecyte does not dispense with any, it enhances the offering within a facility. 

To gain a little insight into cells doing weird things I encourage you to link up with the Phasefocus Twitter feed https://twitter.com/PhaseFocus1

Further to this, please contact us at ATA Scientific. We will be happy to introduce you to some systems, perhaps arrange a demonstration. Call us on +61 2 9541 3500 or send an email to pdavis@atascientific.com.au

References

1. https://www.nationalgeographic.org/encyclopedia/cell-theory/ site accessed 1Feb2022
2. Godin AG, Lounis B, Cognet L. Super-resolution microscopy approaches for live cell imaging. Biophys J. 2014;107(8):1777-1784. doi:10.1016/j.bpj.2014.08.028
3. The Virtual Lens, Phasefocus. https://www.phasefocus.com/technology Website accessed 7 Feb2022.

Livecyte combines high contrast label-free imaging with correlative fluorescence and automatic cell tracking algorithms to provide a complete solution for quantifying phagocyte behaviour down to the single-cell level. This enables users to: 

• Automatically quantify macrophage phenotypic behaviour label-free to measure immune response
• Quantification to the single-cell level gives a more accurate measure of phagocytosis than standard population-level analyses
• Increased accuracy reveals subtle behaviour changes from how phagocyte activity changes over time to saturation

Recently Phasefocus have turned their research to Macrophages, the heroes of our immune response that are commonly misunderstood. Livecyte has uncovered some interesting phenotypes that were in the realms of theory before now! 

What is the macrophage immune response?

Macrophages are effector cells of the innate immune system that phagocytose bacteria and secrete both pro-inflammatory and antimicrobial mediators(1). In addition, macrophages play an important role in eliminating diseased and damaged cells through their programmed cell death. Generally, macrophages ingest and degrade dead cells, debris, tumour cells, and foreign materials, they exist in multiple states of readiness. 

In tissues, they are typically in a “resting” state where they slowly proliferate and clear up debris. 

Macrophage activation occurs when these resting macrophages receive chemical signals which alert them to the proximity of invaders. For example, Lipopolysaccharide (LPS), a component of the outer cell membrane of Gram-negative bacteria such as E. Coli, can be shed by these bacteria, and bind to receptors on the surface of macrophages. In response, these macrophages become activated and produce inflammatory cytokines, reactive oxygen and nitrogen species and begin phagocytosing the foreign bodies. 

What’s the problem we are trying to solve? 

Macrophage immune response is a complex, multifaceted process involving changes to many aspects of macrophage phenotypic behaviour. Current leading applications provide only a population level analysis, using fluorescence expression.

Population level analyses that, for example, look at total fluorescence or pixel area of expression over simplify the response and completely miss much of the richness of phenotypic response. They are also vulnerable to confounding effects from cell proliferation and seeding density variation.

How does Livecyte solve this? 

Livecyte solves this by measuring immune response on a single cell level. This provides a new level of accuracy in phagocytosis quantification and preserves heterogeneity of response. It also brings all the standard benefits of Livecyte to the application such as reduced phototoxicity and measurement of the full breath of phagocyte phenotypic response enabling the correlation of these behaviours and new scientific insights.

In a recent application Note AN018 Macrophage Phagocytosis of Bioparticles, Phasefocus examined the effects upon treating RAW cells with cytochalasin D, an actin inhibitor. Livecyte was used to detect proliferative, and morphological changes to macrophages corresponding with phagocytic activity. Using quantitative, correlative fluorescence detection Livecyte measured changes in rates of phagocytosis of pHrodo Green E. coli Bioparticles.

In this study, the Livecyte Fluorescence Dashboard showed a dose-dependent reduction in the median fluorescence intensity of the cells, therefore reduction in cell phagocytosis, with cytochalasin D treatment. Livecyte’s high content data generation enabled time-lapse data for individual cells to be observed, giving a far more in-depth analysis of an experiment than end point, or total population metrics. Livecyte can observe individual cell behaviour, and therefore can measure fluorescence on a single-cell basis, thus removing assay sensitivity to initial cell count and allowing individual macrophage phagocytosis to be measured independently from cellular proliferation(3).

To summarise the findings of the application AN017 Quantifying Macrophage Activation and Proliferation, Phasefocus characterised subtle changes in macrophage phenotype in response to inflammatory stimuli. Through analysing cell count and cell dry mass values they were able to identify proliferation and growth of cells independently.  A prioritisation towards proinflammatory signalling and growth was identified with an inhibition of the proliferation pathway in cells treated with LPS. This is known to occur in activated macrophages to meet the demands of cell growth and production of bactericidal factors. This was exacerbated with the addition of pro-inflammatory cytokine IFNγ suggesting a synergistic effect of both these pro-inflammatory mediators(2).

In this Application Note, AN019 – Macrophage Phagocytosis of Apoptotic Cells, Livecyte enabled users to reliably investigate time-sensitive changes of immune cells in response to target cells to enhance our understanding of efferocytosis regulation. From a single experiment, it was possible to generate a host of quantitative insights to both substantiate and validate existing data as well as bring new facets to investigate the complex mechanisms that make up a biological response. Livecyte is a crucial tool in bringing a new dimension to traditional in vitro immune assays, advancing the knowledge, and understanding of these pathways(4).

Streamline your work

To truly appreciate the power of the Livecyte, we encourage a much closer scrutiny of these and other applications as you would any scientific paper.

Take a look at what Greg Perry, Image Resource Facility Microscopy Manager at St George’s University of London, has to say. “One professor has been using the Livecyte on a daily basis and his work flow has become so streamlined that he has managed to get a few months’ worth of work done in just one.”

ATA Scientific manages Livecyte throughout Australia and New Zealand. We can arrange virtual and live demonstrations of this amazing microscope. The Livecyte really is the solution to the problem you never knew you had, uncovering novel discoveries using a platform that has transformed other fields, winning multiple awards and breaking Guinness records.  Your research is far too important to miss out on Livecyte, to paraphrase Dr Peter O’Toole of York University – “every microscopy facility should have a Livecyte”.  Contact Peter Davis at ATA Scientific for further information: pdavis@atascientific.com.au.

References

1.       Hirayama D, Iida T, Nakase H. The Phagocytic Function of Macrophage-Enforcing Innate Immunity and Tissue Homeostasis. Int J Mol Sci. 2017;19(1):92. Published 2017 Dec 29. doi:10.3390/ijms19010092

2.       Phasefocus – AN017 – Quantifying Macrophage Activation and Proliferation https://www.phasefocus.com/resources/app-notes

3.       Phasefocus – AN018 – Macrophage Phagocytosis of Bioparticles https://www.phasefocus.com/resources/app-notes

4.       Phasefocus – AN019 – Macrophage Phagocytosis of Apoptotic Cells https://www.phasefocus.com/resources/app-notes

How do you resolve an issue in a product when your analytical methods fail to indicate any difference between batches? Consider the ramifications should your pharmaceutical product cause harm despite your best efforts of your procedures, not to mention the need to analyse in non-native conditions. Most basic assessments cannot delineate conformational effects from colloidal effects . 

Making complex processes simple

Biophysical characterisation of proteins can be a daunting task, given the number of methods required to elucidate the results for Aggregation, Quantitation, Stability, Similarity and Structure. RedShift Bio’s AQS³ pro, powered by Microfluidic Modulation Spectroscopy (MMS), can perform these 5 measurements in a single analysis which is impressive. Sensitivity is improved up to 30 times that of conventional FT/IR instrumentation with a desirable concentration range of 0.1mg/ml to over 200mg/ml and with protein analytics that is equally enviable to the operator. Savings in sample testing time can be more than 80% thanks to the fully automated multi-sample capability.

Often the buffers used in a formulation are not compatible for the analytical method, as seen with spectropolarimetry. The AQS³ pro experiences no interference from excipients in the buffer. This is a game changer, to measure at concentration, and in the final drug conditions, removing the guesswork from formulation and de-risking many steps.

What is the magic behind the AQS³ Pro system?

There are 3 key components in the AQS³ pro that allow the system to achieve all this and separates it from all other systems,

1. a mid IR tuneable quantum cascade laser
2. a thermal, electrically cooled detector
3. a Y shaped microfluidic transmission cell. 

The Tuneable laser provides an optical signal almost 100X brighter than the conventional light source used in FT/IR allowing the use of a simple thermal electrically cooled detector without the need for liquid nitrogen cooling. Given the intensity of the laser, the system is amenable to low concentration samples as low 0.1mg/ml. The measurement differs from the conventional as well. The sample and the reference stream are injected alternately through the Y shaped microfluidic cell passing through the observation zone. Alternating at a rate of 1 – 5hz, the absorbance of the reference and sample are measured almost simultaneously allowing the reference absorbance to be subtracted from the sample absorbance in real time resulting in the collection of reference corrected absorbance spectra. Such a real time buffer subtraction and auto-referencing greatly enhances the sensitivity method and produces an almost drift free signal. 

These innovations create the 30X sensitivity boost. The speed of the AQS³ pro impresses. Where it may take 30 mins per sample for the FT/IR, the AQS³Pro system with MMS technology can measure samples in less than 1.5min. The AQS³ delta analytics packages are refreshingly simple and intuitive, applying advanced analytical tools enabling its use in a vast range of applications across the industry.

What does the AQS³ pro actually measure?

This is an infrared system that measures the Amide 1 band of a protein which is in the wavenumber range of about 1580 – 1720. It takes about 32 data points across the range alternating between buffer and sample, plotting a differential absorbance which is interpolated into a spectrum, which in turn is converted to a second derivative affording fine detail changes from spectrum to spectrum to be elucidated. Flipping the spectra shows the baselines and area of overlap plot enables you to look at similarity and trends between spectra. This can then be deconvoluted using a normal gaussian deconvolution, giving rise to secondary structure motifs, the percentage of the components such as α-Helix, β-Sheet, and Anti-parallel β-Sheet Higher Order Structures (HOS).

Case study: complexity of a vaccine.

The diverse composition of a vaccine makes biophysical characterisation challenging. Not only does a vaccine have antigens and antibodies, they can also have an array of excipients such as preservatives, stabilisers, and buffers plus they can contain antibiotics. The concentrations of the components vary by orders of magnitude and the particle size distribution of a final formulation can range from nano to micron. 

The additional ingredients can introduce protein and non-protein, organic and inorganic materials. If you add up the numbers of tests and instrumentation required to perform these tests it can be incredibly diverse. MMS is a single technology that provides unique insights into many of the parameters required to fully understand these biophysical properties of the sample, from looking at interaction effects of antigen and adjuvant or the effect of looking at varying stabiliser concentrations or altering the buffer pH. All these steps can be performed to understand their effect on the protein secondary structure.

The total characterisation of a protein-based vaccine should include:

Biophysical Characterisation to understand Protein antigen properties such as pH, ionic strength, HOS, and aggregation propensity.

Stabiliser Screening These convey stability by addition of amino acids, surfactants, proteins, sugars and antioxidants to improve shelf life.

Adjuvant Screening Adjuvant surface chemistry (eg alum) as well as adjuvant-antigen interactions should be characterised.

Process Design & Control Process design and vaccine-surface interactions of process equipment should be understood – ie protein loss on filtration substrates as well as denaturation of protein samples.

Stability Studies Real time and accelerated stability studies ensure antigen is in chemically and physically stable state

With this in mind, let’s look at the tools and parameters needed for such biophysical characterisation of protein antigens; from molecular weight in a chromatography system to surface charge on an electrophoretic light scattering system. A few are listed below.

LC – HPLC and SEC Liquid Chromatography in the form of reverse phase, ion exchange, size exclusion can be used to assess chemical and physical stability.

DSC- Differential scanning Calorimetry Thermal stability properties of antigen for insights into formulation conditions.

MALDI – Maldi TOF  High resolution molecular weight providing information about primary structure and post translational modifications.

ELS – Electrophoretic Light Scattering – Surface charge of pure adjuvant versus adjuvant in formulation will identify protein adjuvant interactions.

DLS – Dynamic Light Scattering Particle size distribution of antigen, adjuvant and complex mixtures, Colloidal stability parameters KD and B22

It is important to note – all these parameters need to be measured at some point during development of a protein vaccine.

Where does MMS fit into the development of vaccines? 

Given MMS measures protein secondary structure, we can leverage this capability to measure in final formulation conditions to assess HOS as stabilisers are varied to determine if they impart a stabilising effect and prevent aggregation processes. The AQS³ pro could be used for adjuvant screening, investigating the interaction and to determine if it is damaging to the antigen. Process design can be studied to establish, for example, if a filtration membrane causes a negative effect on the structure of the antigen, or to understand the stability of the formulation using long-term stability studies. It is now clear that MMS can be applied across the entire development pipeline with a wide range of measurements to help understand the full properties of protein-based vaccines.

De-Risk Drug Development

MMS’s sensitive, accurate measurements coupled with a robust data analysis package provides simple, accessible, and reliable results to de-risk and accelerate drug development workflows. The AQS³ pro can identify at-risk candidates far earlier than traditional methods, clearly saving time and resources.

ATA Scientific are proud to have the RedShiftBio AQS³ pro within our suite of instrumentation. Should you require further information on the AQS³ pro or indeed many of the techniques cited above, please do not hesitate to contact us.

The recent approval of COVID vaccines from Moderna and Pfizer based on mRNA-containing lipid nanoparticles (LNPs) has propelled this pioneering technology, shifting it from being simply viewed as speculative research to becoming transformative in the area of genetic medicines. The pharmaceutical world has seen vaccine development experience a sharp jolt, evolving from the 1950’s concept of one egg, one vaccine dose and the bulk cellular expansion in bioreactors, to a highly efficient and timely manufacturing protocol. The advent of mRNA containing LNPs has enabled a highly effective new vaccine platform, but at the same time has raised many questions.

Why has this new technology changed our dependence on cell cultured vaccines? Will the NanoAssemblr democratise vaccine production globally?

The 4 pillars of RNA vaccine development

Classically there are four pillars of vaccine development, individually they are inconsequential; but together they can be a formidable assault on pathogenic viral invaders! These pillars are Antigens, Vectors, Delivery-Nanoparticles, and Manufacturing. Each pillar has its set of design and development considerations and associated technologies.

To streamline understanding of each pillar it is important to explore these further details; an Antigen is what the body defences identify as foreign and will attack. In the case of COVID much of the focus has been on the characteristic spike protein. Some of the vaccines are designed to create a copy of this passive component of the virus to educate the immune system to respond by producing antibodies. A viral vector is generally a harmless virus (often an Adenovirus) that is hijacked to carry the genetic code of interest (ie the spike protein) into the cells, like a letter is placed into an envelope to be posted. In this discussion consider the Vector as a replicon virus such as an Alphavirus where the structural protein code is substituted for the Gene Of Interest (GOI), being the spike protein, but retaining the four non-specific proteins (NSP) that work together to actively replicate the GOI once in the ribosome – ultimately producing a huge amount of antigens, a self-amplified RNA ¹. A bit of RNA code is quite vulnerable, your body is trained to destroy free floating RNA, so it needs to catch an uber to enter the body and ultimately the cells. This uber needs to be able to navigate around our defences. To do this, a suitable camouflage is needed.

A Lipid Nanoparticle (LNP) is perfect to encapsulate the code thereby becoming the delivery pillar and it has huge scalable potential for manufacturing, which is the next pillar. Whilst it is a challenge to bring all these components together, the ultimate challenge is to manufacture to scale. Translating research to usable medicine is often a bottleneck and many candidates fall over at this stage. It is exceptionally important to de-risk the process well before this stage. This is where the Precision Nanosystems platform products – the NanoAssemblr range – have had a huge impact in translating research to the clinic.

The anatomy of lipid nanoparticles (LNP)

Often the terms Liposome and LNP are used interchangeably, however, whilst they are similar in many ways, there are distinct differences in their function and structure. Consider a Liposome is made up of a Lipid bilayer primarily composed of amphipathic phospholipid enclosing an interior aqueous space, it can be decorated with a protein adding targeted delivery to its capabilities. LNPs can take on a variety of forms enhancing their ability to encapsulate an assortment of cargoes like peptides, genetic payloads like siRNA, mRNA and saRNA plus other small molecules.

The most exciting of these are those formulated with ionisable cationic lipids.  Recently research from Meng and Grimm proposes LNPs composed of the best-performing iPhos and different helper lipids—zwitterionic lipids, ionizable cationic lipids and permanently cationic lipids—achieved selective organ targeting (SORT) and organ-specific CRISPR-Cas9 gene editing in spleen, liver, and lungs of mice, respectively³ .

Mechanisms of LNP action and the role of different lipid components

The LNP structure paves the way for a nanoprecipitation method for their creation.  Precision Nanosystems employ a microfluidic architecture that allows two streams to mix under laminar flow conditions enabling the process to be reproducible and scalable. Importantly this mixing rate is rapid, <1 millisecond adhering to the tenent ‘the mixing rate should be faster than assembly rate’.

Consider one of these streams is aqueous, a buffer, such as PBS to maintain the pH whilst carrying the payload. The other stream has the lipid components dissolved in a solvent such as ethanol. Controlling the flow rate ratios and the total flow rate will vary the nanoparticle size. A LNP is not a solid sealed system like a rubber ball, instead it is a collection of elements that are bound by charge. In a mRNA example, the cationic lipids meet the negative charges of the phosphate backbone of the mRNA. Then the neutral helper lipids such as zwitterionic lipids stabilise the lipid bilayer of the LNP. This enhances delivery efficiency, and thanks to the aqueous environment, polyethylene glycol (PEG)-lipid forms the outer shell pointing it’s hydrophobic tails inwards. These improve the colloidal stability in biological environments by reducing specific absorption of plasma proteins and forming a hydration layer over the nanoparticles⁴. The result is unilamellar, predictably sized, uniform LNPs created in a matter of seconds. 

Precision Nanosystems’ NanoAssemblr series facilitates the encapsulation of genetic material into ionisable cationic lipids ideal to be seen as ‘self’ by the body – moving by stealth into the cells by endocytosis. The cellular uptake of LNP mainly relies on the endocytic pathway. More in detail, it has been shown that specific serum proteins adsorbed on the surface of LNPs upon intravenous injection can drive the cell internalisation⁵. For an effective nucleic acid delivery, a large portion of functional molecules should escape the endosomal compartment before the degradation cascade begins. Ionizable lipids, which are capable of modulating their charge depending on the environmental pH, are recognised as a key component of LNPs for the endosomal escape⁶.

NanoAssemblr for vaccine development

Lipid nanoparticles (LNPs) are the most clinically advanced non-viral gene delivery system. Lipid nanoparticles safely and effectively deliver nucleic acids, overcoming a major barrier preventing the development and use of genetic medicines and vaccines. The Precision Nanosystems platform facilitates the formulation of LNP vaccines on a research scale through to full GMP manufacture. The nanoparticles produced work better than other methods of manufacture such as T-Tube mixing, in a study siRNA-LNPs manufactured by three NanoAssemblr® instruments exhibited encapsulation efficiencies of higher than 95%, Factor VII siRNA knockdown efficacy was maintained for nanoparticles produced on the NanoAssemblr® Benchtop, Blaze, and GMP System. 

Particles generated by the NanoAssemblr® platform are more uniform than those made by conventional T-Tube mixing methods. T-tube generated lipid nanoparticles exhibit a multilamellar morphology vs the homogeneous-core structure for the NanoAssemblr® generated lipid nanoparticles. Serum Factor VII siRNA knockdown efficacy was higher for NanoAssemblr® siRNA lipid nanoparticles compared to conventional T-tube lipid nanoparticles, 72 hours following systemic administration⁷.

Your nano solution

ATA Scientific are just one call away to demonstrate the rapid, effortless lipid nanoparticle production, and optimisation. We can tailor the system choice to suit your stage of development.

ATA Scientific have a portfolio of products that complement the NanoAssemblr range such as the Malvern Panalytical Zetasizer, ITC and Nanosight plus microscopy and cell counting solutions. Contact us today for an illuminating discussion. 

REFERENCES:

1)      Self-Amplified RNA Vaccine Against COVID-19.   https://youtu.be/tVh1s06H_nw

2)      Liposomes vs. Lipid Nanoparticles: Which Is Best for Drug Delivery? https://www.biochempeg.com/article/122.html Accessed 24 Sept 2021

3)      Meng, N., Grimm, D. Membrane-destabilizing ionizable phospholipids: Novel components for organ-selective mRNA delivery and CRISPR–Cas gene editing. Sig Transduct Target Ther 6, 206 (2021). https://doi.org/10.1038/s41392-021-00642-z

4)      Cullis, P. R., and Hope, M. J. (2017). Lipid nanoparticle systems for enabling gene therapies. Mol. Ther. 25, 1467–1475. doi: 10.1016/j.ymthe.2017.03.013

5)      Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 2020;15:313-320. https://doi.org/10.1038/s41565-020-0669-6.

6)      Schlich, M, Palomba, R, Costabile, G, et al. Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles. Bioeng Transl Med. 2021; 6:e10213. https://doi.org/10.1002/btm2.102137)      https://www.precisionnanosystems.com/workflows/formulations/lipid-nanoparticles