Application Note

Lipid Nanoparticle Payload Capabilities

Lipid nanoparticles (LNPs) have emerged as one of the most versatile and clinically successful drug delivery platforms, revolutionizing therapeutic applications ranging from mRNA vaccines to gene editing systems. Their unique structure and composition enable the encapsulation and targeted delivery of a remarkably diverse range of therapeutic payloads, making them a cornerstone technology in modern nanomedicine.

Fig 1. Types of payloads delivered by LNPs with their applications in drug delivery.

1. Overview of LNP Structure and Functionality

LNPs are sophisticated delivery vehicles composed of four key lipid components: ionizable cationic lipids, helper phospholipids, cholesterol, and PEGylated lipids. The ionizable lipids are particularly crucial, as they can switch from neutral at physiological pH to positively charged at low pH, facilitating both cargo encapsulation during formulation and endosomal release within target cells. This pH-responsive behavior enables LNPs to protect their cargo during circulation while efficiently releasing it intracellularly.

The assembly mechanism of LNPs involves rapid mixing of an aqueous phase (containing the therapeutic cargo) with an organic phase (containing the lipid mixture.) This triggers self-assembly of stable nanoparticles typically ranging from 80-200 nm in diameter. The resulting particles have a complex internal structure where ionizable lipids entrap negatively charged nucleic acids in the core, surrounded by a lipid shell containing helper lipids, cholesterol, and PEGylated components.

Fig 2. LNP synthesis with microfluidic mixing.

2. Nucleic Acid Payloads

2.1 mRNA

Messenger RNA (mRNA) represents the most common and clinically advanced payload for LNPs, exemplified by the COVID-19 vaccines that have demonstrated global efficacy and safety. LNPs protect mRNA from degradation by serum nucleases and facilitate its intracellular delivery, where it undergoes translation to produce the encoded protein.

Fig 3. Recent advances in mRNA-LNP therapeutics. Image adapted from Journal of nanobiotechnology. 2022 Jun 14;20(1):276.

Recent single-particle analysis revealed that benchmark mRNA LNP formulations typically contain 2-3 mRNA molecules per loaded particle, with 40-80% of particles being empty depending on assembly conditions. Remarkably, payload capacity correlates with mRNA mass rather than copy number, meaning smaller mRNAs can achieve higher copy numbers per particle.

Some clinical applications of mRNA-LNPs include:

Vaccines: COVID-19 vaccines (Pfizer-BioNTech, Moderna), with expanding applications to other infectious diseases and cancer immunotherapy
Protein replacement therapy: Delivering mRNA encoding therapeutic proteins for genetic diseases
Cancer therapy: mRNA encoding tumor antigens, cytokines (IL-21, IL-7), or immune modulators

2.2 siRNA

Small interfering RNA (siRNA) is one alternative to mRNA. siRNA-LNPs have achieved FDA approval with Onpattro (patisiran) for treating hereditary transthyretin-mediated amyloidosis. These smaller nucleic acids (~21 base pairs) show different packaging characteristics compared to mRNA, with studies indicating no empty LNPs in siRNA formulations.

Fig 4. Schematic representation of mechanism of LNP-mediated siRNA delivery into cells resulting in gene silencing. Image adapted from Pharmaceutics. 2022 Nov 19;14(11):2520.

Recent single-particle analysis revealed that benchmark mRNA LNP formulations typically contain 2-3 mRNA molecules per loaded particle, with 40-80% of particles being empty depending on assembly conditions. Remarkably, payload capacity correlates with mRNA mass rather than copy number, meaning smaller mRNAs can achieve higher copy numbers per particle.

Some clinical applications of siRNA-LNPs include:

Genetic diseases: Targeting specific disease-causing genes in the liver
Cancer therapy: Silencing oncogenes such as Bcl-2, MYC, or growth factor receptors
Metabolic disorders: Targeting cholesterol metabolism genes like PCSK9

2.3 Gene Editing Systems

LNPs enable reliable delivery of CRISPR-Cas9 components, whether as mRNA precursors or preformed ribonucleoprotein (RNP) complexes.

mRNA Delivery: This method involves co-encapsulation of Cas9 mRNA and guide RNA. This may be done in either single or separate particles, and can achieve median editing rates of 38.5% in liver hepatocytes.

Fig 5. Schematic illustration of LNP-CRISPR. Image adapted from Nature communications. 2021 Dec 8;12(1):7101.

RNP Delivery: This method involves direct delivery of preformed Cas9-gRNA complexes. Some advantages include reduced off-target effects, improved editing efficiency, and shorter intracellular half-life. Recent advances using thermostable genome editors enable robust RNP encapsulation and tissue-selective editing in both liver and lung tissues.

Fig 5. Schematic illustration of LNP-CRISPR. Image adapted from Nature communications. 2021 Dec 8;12(1):7101.

2.4 circRNA

Circular RNAs (CircRNA) represent a paradigm shift in RNA therapeutics due to their unique covalently closed-loop structure that lacks the 5' and 3' ends found in linear RNA. This circular topology confers remarkable stability against exonuclease degradation, as these enzymes typically require free RNA ends to initiate cleavage. Endogenously produced circRNAs demonstrate 2-5 times greater stability compared to their linear counterparts, with potential therapeutic applications benefiting from this enhanced durability.

CircRNA vaccines have demonstrated robust immunogenicity in preclinical models, with mannose-modified LNP-circRNA vaccines showing comparable immune responses to conventional platforms in both rabies virus and SARS-CoV-2 models. The enhanced stability profile enables room temperature storage after lyophilization, addressing critical cold-chain limitations of current mRNA vaccines.

Fig 7. Transfection with Circular RNA. Image adapted from MBio, 15(1), pp.e01775-23.

2.5 SaRNA

Self-amplifying RNAs (SaRNA) are considerably larger molecules (9-12 kb) compared to conventional mRNA, containing the basic mRNA elements (cap, 5' UTR, 3' UTR, poly(A) tail) plus a substantial open reading frame encoding four non-structural proteins (nsP1-4) and a subgenomic promoter. Upon cellular delivery, saRNA undergoes translation to produce the four components of an RNA-dependent RNA polymerase (RDRP) complex. This RDRP complex, tethered to plasma membrane invaginations, synthesizes complementary negative-strand RNA, which subsequently serves as a template for producing both genomic and subgenomic positive-strand RNA in excess. The self-amplifying nature enables sustained protein expression for 20-26 days compared to 2-3 days for conventional mRNA.

Multiple saRNA vaccine candidates have advanced to clinical trials, including GEMCOVAC-OM (Gennova Biopharmaceuticals) and ARCT-154, both targeting SARS-CoV-2. The KOSTAIVE (ARCT-154) saRNA COVID-19 vaccine has shown promising safety and efficacy profiles in Phase I/II trials. Recent clinical data demonstrate that optimized saRNA vaccines can achieve protective immunity at ultra-low doses, with some formulations effective at doses 100-1000 times lower than conventional mRNA vaccines.

Fig 8. Transfection with SaRNA. Image adapted from Scientific Reports, 11(1), p.21308.

2.6 Other Nucleic Acids

LNPs can also successfully deliver various other nucleic acid payloads.

DNA: For longer-term gene expression and gene therapy applications

Fig 9. Corosolic acid-modified lipid nanoparticles as delivery carriers for DNA vaccines against avian influenza. Image adapted from International Journal of Pharmaceutics. 2023 May 10;638:122914.

Antisense oligonucleotides (ASOs): Short sequences (10-30 nucleotides) for modulating gene expression
microRNA mimics and inhibitors: For regulating cellular pathways

3. Proteins and Peptides

3.1 Antibodies and Immunomodulatory Proteins

LNPs can encapsulate and deliver various proteins, though this represents a more challenging application than nucleic acid encapsulation. One major concern is protein stability in the typical organic solvents used during formulation. Nevertheless, recent advances have enabled delivery of:

Fig 4. Schematic representation of mechanism of LNP-mediated siRNA delivery into cells resulting in gene silencing. Image adapted from Pharmaceutics. 2022 Nov 19;14(11):2520.

Therapeutic antibodies: For targeted cancer therapy and immunomodulation
Cytokines and growth factors: Including interferon and interleukins for cancer immunotherapy
Enzyme replacement: For treating genetic enzyme deficiencies

3.2 Peptide-Based Degraders

An emerging application involves delivery of peptide-based proteolysis targeting chimeras (PepTACs), which can degrade specific intracellular proteins. These amphiphilic peptides require careful design optimization, with longer hydrophobic tails showing improved LNP encapsulation and cellular delivery.

Fig 11. Encapsulation of PepTACs in LNPs allows for endosomal uptake, escape, and cytosolic delivery. Image adapted from bioRxiv. 2024 Mar 19:2024-03.

4. Small Molecule Drugs

LNPs excel at delivering hydrophobic small molecule drugs. The lipid bilayer structure provides an ideal environment for encapsulating poorly water-soluble chemotherapeutics. Notable examples include:

Doxil®: This drug is a stealth liposome encapsulating doxorubicin. It is used for the clinical treatment of ovarian and metastatic breast cancer as well as various forms of myeloma. Because of the encapsulation of doxorubicin inside PEGylated liposomes, the side effects of free doxorubicin, including chronic cardiomyopathy and congestive heart failure, were significantly mitigated. In addition, the PEGylation of liposomes in Doxil® supported prolonging the circulation time of this formula after administration.

Fig 12. Structure of FDA approved Doxil®. Image adapted from Vaccines. 2021 Apr 8;9(4):359.

Combination Therapies: In more general examples, small molecules can be conjugated to siRNA within LNPs for combination chemotherapy and RNA interference therapy, showing enhanced anti-tumor efficacy in lymphoma models.

Fig 13. Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy. Image adapted from Acta Pharmaceutica Sinica B. 2023 Apr 1;13(4):1429-37.

5. Payload Capacity and Size Limitations

The encapsulation efficiency and payload capacity of LNPs depend on several critical factors:

5.1 Size-Dependent Packaging

Research reveals that LNP payload capacity correlates with particle mass rather than molecular count. For nucleic acids:

mRNA (1000-2000 nucleotides): Typically 2-3 molecules per loaded LNP
siRNA (~21 base pairs): Higher copy numbers achievable due to smaller size
Larger mRNA or DNA constructs: May result in higher percentages of empty particles

5.2 Payload Distribution Heterogeneity

Single-particle analysis reveals significant heterogeneity in payload distribution, with 40-80% of particles potentially being empty in some formulations. This heterogeneity arises from the balance between RNA diffusion kinetics and lipid self-assembly dynamics during formulation.

5.3 Optimization Strategies

Several approaches can improve payload uniformity and capacity:

Controlled mixing conditions: Turbulent mixing minimizes payload variance
Salt concentration optimization: Systematic adjustment enhances RNA loading
PEG lipid content: Fine-tuning affects both particle size and payload capacity

LNPs have established themselves as a transformative drug delivery platform with the versatility to accommodate an extraordinary range of therapeutic payloads. From the clinical success of mRNA vaccines to emerging applications in gene editing and cancer therapy, LNPs continue to expand the boundaries of what is possible in targeted drug delivery. As the technology matures, ongoing research focuses on optimizing payload capacity, enhancing targeting precision, and ensuring long-term safety for diverse therapeutic applications. The future of LNP-based therapeutics appears exceptionally promising, with the potential to revolutionize treatment paradigms across numerous disease areas.

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Application Note

Lipid Nanoparticle Payload Capabilities

Fig 1. Types of payloads delivered by LNPs with their applications in drug delivery.

1. Overview of LNP Structure and Functionality

Fig 2. LNP synthesis with microfluidic mixing.

2. Nucleic Acid Payloads

2.1 mRNA

Fig 3. Recent advances in mRNA-LNP therapeutics. Image adapted from Journal of nanobiotechnology. 2022 Jun 14;20(1):276.

2.2 siRNA

Fig 4. Schematic representation of mechanism of LNP-mediated siRNA delivery into cells resulting in gene silencing. Image adapted from Pharmaceutics. 2022 Nov 19;14(11):2520.

2.3 Gene Editing Systems

Fig 5. Schematic illustration of LNP-CRISPR. Image adapted from Nature communications. 2021 Dec 8;12(1):7101.

Fig 5. Schematic illustration of LNP-CRISPR. Image adapted from Nature communications. 2021 Dec 8;12(1):7101.

2.4 circRNA

Fig 7. Transfection with Circular RNA. Image adapted from MBio, 15(1), pp.e01775-23.

2.5 SaRNA

Fig 8. Transfection with SaRNA. Image adapted from Scientific Reports, 11(1), p.21308.

2.6 Other Nucleic Acids

Fig 9. Corosolic acid-modified lipid nanoparticles as delivery carriers for DNA vaccines against avian influenza. Image adapted from International Journal of Pharmaceutics. 2023 May 10;638:122914.

3. Proteins and Peptides

3.1 Antibodies and Immunomodulatory Proteins

Fig 4. Schematic representation of mechanism of LNP-mediated siRNA delivery into cells resulting in gene silencing. Image adapted from Pharmaceutics. 2022 Nov 19;14(11):2520.

3.2 Peptide-Based Degraders

Fig 11. Encapsulation of PepTACs in LNPs allows for endosomal uptake, escape, and cytosolic delivery. Image adapted from bioRxiv. 2024 Mar 19:2024-03.

4. Small Molecule Drugs

Fig 12. Structure of FDA approved Doxil®. Image adapted from Vaccines. 2021 Apr 8;9(4):359.

Fig 13. Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy. Image adapted from Acta Pharmaceutica Sinica B. 2023 Apr 1;13(4):1429-37.

5. Payload Capacity and Size Limitations

5.1 Size-Dependent Packaging

5.2 Payload Distribution Heterogeneity

5.3 Optimization Strategies

References:

Related Resources

Application Notes

Publications

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