Supramolecular Pharmacy 10. Nanoparticles for drug delivery Ondřej Jurček 1 Nanochemistry, nanotechnology, nanoparticles 2 • Chemistry of multi-nanometer scale molecules (dimensions of 2-200 nm) • Nanotechnology a field of applied science and technology based on the control, manipulation, and fabrication of matter on the size scale approaching that of atoms and molecules • Nanotechnology is highly multidisciplinary and draws on techniques and knowledge from applied physics, materials science, interface and colloid science, device physics, chemical and biological engineering and of course supramolecular chemistry • Nanoparticles (NPs) very small usually approximately spherical fragments of material of radius in the approximate range 2–100 nm • Nanoparticles can be made of any material and includes crystalline or amorphous solid particles • NPs = nanocrystals, quantum dots (semiconductor particles having often fluorescence), nanoclusters (1-10 nm NPs) • NPs are usually monodispersed (size distribution <15 %) • NPs are often used as suspension in some kind of solvent (colloid) • Solid usually does not separate as a precipitate (size, stabilization by coating – prevents aggregation, increases solubility) Nanoparticles (NPs) in drug delivery (nanocarriers) 3 • Oral, local, topical, and systemic nano-/microparticle delivery systems have been approved by FDA or EMA • Intravenously administered NPs are the most studied preclinically and clinically • Systemic delivery provides direct access to nearly all parts of the body • Organic (including polymeric micelles and vesicles, liposomes, dendrimers, and hydrogels) and inorganic (including quantum dots, gold and mesoporous silica NPs) • Organic materials: (a) NPs for gene therapy applications or (b) NPs for delivery of small molecule drugs for cancer treatment • Organic NPs provide enhanced drug protection, controlled release, extended circulation, improved targeting to diseased tissues/cells • Inorganic NPs provide the same and stimuli responsive functions due to surface plasmon resonance (thermal heating or imaging) or magnetic responsiveness (MRI, magnetic targeting) • Introducing new or improving the old therapies Anselmo et al. Bioengineering & Translational Medicine 2016, 1, 10-29. Clinically relevant NPs and their applications 4Anselmo et al. Bioengineering & Translational Medicine 2016, 1, 10-29. Black: approved applications, Red: Application in clinical trials (in 2016). • Nanoparticles have been developed to overcome the limitations of free therapeutics and navigate biological barriers — systemic, microenvironmental, and cellular — that are heterogeneous across patient populations and diseases • Personalized delivery – precision medicine Organized efforts towards precision medicine 5 • US National Science and Technology Council (NSTC) launched the National Nanotechnology Initiative (NNI) in 2000 • The number of nanomedicines available to patients is drastically below projections for the field • Gap comes from a lack of understanding of the differences in physiology and pathology between animal model species and humans • Heterogeneity amongst patients can also limit the success of nanomedicines • Few are recommended as first-line treatment options, and many show improvements in only a small subset of patients • Moreover, growth, structure and physiology of diseased tissue alter NP distribution and functionality • Complex systems: nanocarrier-mediated combination therapies — to alter multiple pathways, maximize the therapeutic efficacy against specific macromolecules, target particular phases of the cell cycle or overcome mechanisms of drug resistance Mitchell et al. Nature Reviews 2021, 20, 101–124. Organized efforts towards precision medicine 6 • New focus on generating NPs to overcome biological barriers specific to patient subsets or disease states can be attributed, in part, to the increasing prevalence of precision, or personalized medicine and the creation of the Precision Medicine Initiative (PMI) in 2015 • Precision medicine is to utilize patient information — such as genetic profile, environmental exposures or comorbidities — to develop an individualized treatment plan • Minimizes the impact of patient heterogeneity and allows for more accurate patient stratification, improved drug specificity and optimized dosing or combinatorial strategies Mitchell et al. Nature Reviews 2021, 20, 101–124. Biological barriers to precision medicine applications 7 * Plasma proteins that induce inflammation and aid in the clearance of foreign bodies or damaged cells by enhancing antibody and phagocytic cell activity • Biodistribution and drug delivery are difficult to achieve as NPs face both physical and biological barriers: shear forces, protein adsorption and rapid clearance • These are often altered in disease states and can be even more difficult to overcome with a generalized, one-size-fits-all approach, but also patient specific • Systemic delivery – circulation, stability, and clearance • While in circulation excretion, blood flow, coronas and phagocytic cells can reduce NP stability and delivery • NPs with a diameter less than 10 nm have generally been shown to be rapidly eliminated by the kidneys, whereas NPs larger than 200 nm risk activating the complement system* • To avoid rapid excretion based on surface properties, many NP formulations incorporate PEG as a stealth coating • PEGylation improves the circulation time by altering the NP size and solubility while shielding the NP surface from enzymes and antibodies that may induce degradation, secretion and clearance Biological barriers to precision medicine applications 8 • Platelet (thrombocyte) membrane cloaking • NP stability is greatly affected by how its composition material interacts with the environment • Lipid-based and polymer-based NPs are the most susceptible to instability and aggregation both in circulation and in storage • The balance between stabilization and effective intracellular delivery — which typically requires carrier degradation — must be considered Mitchell et al. Nature Reviews 2021, 20, 101–124. NPs interaction with blood vessels and blood components 9 • In the bloodstream, NPs experience varying flow rates that induce shear stress which can lead to their damage, prevent their movement into surrounding tissues • Larger microparticles have higher probability of localizing to vessel walls, nonspherical NPs (ellipsoids, disc, and rod-like) show better margination than spherical • Non-specific adherence of serum proteins and lipids forms a corona on the surface of NPs • This corona will dictate the distribution of the NP, and can compromise stability of both the NP and its cargo • For example, coronas containing apolipoprotein E (ApoE) act as targeting moieties for low-density lipoprotein receptors, which leads to NP delivery to hepatocytes and, in some instances, across the blood–brain barrier (BBB) Mitchell et al. Nature Reviews 2021, 20, 101–124. NPs interaction with blood vessels and blood components 10 • Clearance usually results from the interaction with the mononuclear phagocytic system (MPS) or reticuloendothelial system (RES) • Systems feature phagocytes (predominantly macrophages), monocytes and dendritic cells, which take up NPs and accumulate in the spleen and liver • Stiffer NPs are often cleared more rapidly • Cationic NPs are generally most rapidly cleared, followed by anionic NPs, whereas neutral and slightly negative NPs have the longest half-lives in circulation (PEGylation or cell membrane coating prevents clearance) • Spherical NPs induce a stronger immune response overall. Uptake by phagocytic cells has been related to the NP curvature and aspect ratio: triangular and rodshaped NPs show more uptake than star-shaped or spherical NPs, and rodshaped NPs induce more inflammation in macrophages Mitchell et al. Nature Reviews 2021, 20, 101–124. NPs interaction with blood vessels and blood components 11Mitchell et al. Nature Reviews 2021, 20, 101–124. Biological barriers to precision medicine applications 12Mitchell et al. Nature Reviews 2021, 20, 101–124. CAR, chimeric antigen receptor; EGFR, epidermal growth factor receptor; EPR, enhanced permeation and retention; gRNA, guide RNA; RNP, ribonucleoprotein. Enhanced permeability and retention (EPR) effect 13Xie et al. Curr. Cancer Drug Target 2019, 19(4), 257. • high–molecular weight nontargeted drugs and prodrugs accumulate in tissues that offer increased vascular permeability, such as in sites of inflammation or cancer Nanoparticle targeting 14 Mitchell et al. Nature Reviews 2021, 20, 101–124. Tang et al. Nanoscale 2018, 10, 17265-17274. • Many NP platforms have added targeting moieties to their surface to direct their delivery • Targeting moieties: antibodies, glucose, transferrin, folate, transporters and integrin ligands • Use interactions with molecules on the target cell’s surface, such as ligand– receptor, enzyme–substrate or antibody–antigen mediated interactions. • Targeted NPs must be engineered with a targeting moiety density that allows for these cell surface interactions • Ratio of receptors to ligands and the number of interactions needed to overcome the initial energy barrier to NP uptake Common uptake pathways that determine NP fate 15Mitchell et al. Nature Reviews 2021, 20, 101–124. Mode of action 16Kashkooli et al. Journal of Controlled Release 2020, 327, 316–349. FDA/EMA approved (1995/1996) doxil (caelyx) – doxorubicin Ovarian cancer (secondary to platinum based therapies) Oral drug delivery 17Mitchell et al. Nature Reviews 2021, 20, 101–124. • Gastrointestinal tract presents numerous barriers for NPs • Average optimal reported size for NP transcytosis in gastrointestinal applications seems to be around 100 nm • Rod-shaped NPs generally outperform spherical particles • Even when NPs are internalized by intestinal epithelial cells, only a small percentage undergo exocytosis • Transferrin pathway can be exploited for transepithelial movement in the intestine • Problem is nevertheless with formation of coronas • Barriers are made heterogeneous by pathologies, such as inflammatory diseases, that may increase epithelial permeability and alter mucus production, pH and the gastrointestinal microbiome Order in liquids 18 • Biological cell membranes are made of bulky phospholipids that are forced to order themselves in water by combination of hydrophobic effects and interactions between the solvent and the hydrophilic head group, forming capsule-like vesicles – cells • Cell membrane must exist in the liquid phase to allow rapid biochemical transport and solution chemistry • Solid-like long-term structure is needed to preserve membrane potentials, control cellular signaling and protect sensitive intracellular enzymes • Cell membrane must also be sufficiently ordered to anchor transmembrane proteins such as ATPases • Fluid mosaic model Surfactants, micelles, vesicles 19 • Surfactant is a molecule that has two different moieties – polar, hydrophilic and nonpolar lipophilic – they are amphiphiles • Daily components – part of soap, washing liquid Langmuir through Langmuir-Blodgett technique Ordered surfactant structures 20 • The mode of behavior depends on concentration and structure of surfactant • Critical micelle concentration - CMC (T, mol dm-3) Jonathan W. Steed, Jerry L. Atwood for Supramolecular Chemistry, ISBN: 978-1-119-58251-9. Changes of micelles 21 • Micelles go through several changes based on their concentration and size of the surfactant Jonathan W. Steed, Jerry L. Atwood for Supramolecular Chemistry, ISBN: 978-1-119-58251-9. Bilayers and vesicles 22 TEM: 79 000 x – ca 200 nm • Sheet like micelles – vesicles • Cells are formed of phospholipid bilayers • Source of the origin of the first biological cells? • Protocells Liposomes 23Guimarães et al. International Journal of Pharmaceutics 2021, 601, 120571. 20-100 nm >100 nm >500 nm >1000 nm Building units Large et al. Advanced Drug Delivery Reviews 2021, 176, 113851. Tuning their properties Large et al. Advanced Drug Delivery Reviews 2021, 176, 113851. 1. Mix POPC and cholesterol 2. Evaporate solvents 3. Re-dissolve in aqueous soln. of drug 4. Stir for 1 h (vesicle formation) 5. Freeze-thawing (breaking down multilamellar vesicles) or sonication 6. Sizing (formation of vesicles smaller than 200 nm, extruder) 7. Size exclusion separation (removal of small vesicles and free dye) Vesicle preparation – thin film hydration 6. 7. Anion (chloride) transport study POPC Vesicle preparation • Reverse-phase evaporation – LUV, MLV – protein encapsulation • Injection – LUV – many variations, injection of lipids in low boiling solvent • Detergent removal – LUV – surfactant with high CMC • Dehydration-rehydration – LUV – without organic solvent • Etc. • Freeze-thaw - solution can be sonicated at room temperature, frozen in liquid nitrogen and left at room temperature to melt. As the liposomal solution thaws, vesicles fuse forming LUVs. Up to 10 freeze-thaw cycles may be used to achieve the intended results. Large et al. Advanced Drug Delivery Reviews 2021, 176, 113851. Vesicle characterization • Zeta potential = overal net charge of the particles - net charge of liposomes is influenced by key parameters, such as lipid composition, the head group of lipids and associated ligands Guimarães et al. International Journal of Pharmaceutics 2021, 601, 120571. Lipid-based NPs - liposomes 30a) Large et al. Advanced Drug Delivery Reviews 2021, 176, 113851; b) Mitchell et al. Nature Reviews 2021, 20, 101–124. • Lipid-based NPs are the most common class of FDA-approved nanomedicines • Many advantages including formulation simplicity, self- assembly, biocompatibility, high bioavailability, ability to carry large payloads and a range of physicochemical properties that can be controlled to modulate their biological characteristics • Unique physiological functions, such as pH response, prolonged blood circulation, and reduced systemic toxicity • Liposomes are typically composed of phospholipids, which can form unilamellar and multilamellar vesicular structures, usually spherical • Liposomes can carry and deliver hydrophilic, hydrophobic and lipophilic drugs, and they can even entrap hydrophilic and lipophilic compounds in the same system • In vitro and in vivo stability are altered by NP size, surface charge, lipid composition, number of lamellae and surface modifications (with ligands or polymers) • Shelf-stability of liposomes can be increased by freeze-drying Lipid-based NPs – lipid nanoparticles (LNPs) 31 • Lipid nanoparticles (LNPs) - liposome-like structures widely used for the delivery of nucleic acids (form micellar structures within the particle core) • Composition: cationic or ionizable lipids that complex with negatively charged genetic material and aid endosomal escape, phospholipids for particle structure, cholesterol for stability and membrane fusion, and PEGylated lipids to improve stability and circulation • Important in personalized genetic therapy applications for their efficacy of their nucleic acid delivery along with their simple synthesis, small size and serum stability • Ionizable LNPs are an ideal platform for the delivery of these nucleic acid therapies as they have a near-neutral charge at physiological pH but become charged in acidic endosomal compartments, promoting endosomal escape for intracellular delivery • Limited by low drug loading and biodistribution that results in high uptake to the liver and spleen Mitchell et al. Nature Reviews 2021, 20, 101–124. Technological challenges in use of NPs 32 • Scale-up synthesis – it is essential to have consistent and highly reproducible formulation prior to the clinical trials • Performance optimization • Performance prediction – correlation between animal and human model is essential • Sterilization – provide compromise between stability and sterilization 33Anselmo and Mitragotri Bioeng. Transl. Med. 2019, 4, e10143. 34Anselmo and Mitragotri Bioeng. Transl. Med. 2019, 4, e10143. Marketed liposomes Large et al. Advanced Drug Delivery Reviews 2021, 176, 113851. In the next class… Nanoparticles for drug delivery 2 Thank you for your attention!