administration, leading to excellent gene silencing capabilities., - Lipid nanoparticle delivery systems for siRNA-based therapeutics | Wan C, Allen TM, Cullis PR 5. FAQs 2022 MolecularCloud, Reply Emerging Research and Clinical Development Trends of Liposome and Lipid Nanoparticle Drug Delivery Systems. Their biocompatibility results in minimal adverse reactions. Even within a single type of cancer, tumor types differ from one patient to another, and understanding. First, they can shield a drug from detection by the bodys immune system, mimicking biological membranes and giving the drug more time to reach its intended destination. This can result in loss of the encapsulated drug or unfavorable mixing of different vesicles cargo. LNPs also provide mechanical stability, controlled morphology and narrow size distribution.1, Inorganic materials, organic materials and hydrogels have each been explored as cores for liposomal nanoparticles, encapsulated within varying numbers of lipid layers that form the shell. Various strategies have been employed to design conventional liposomes with triggered-release capabilities, enhancing therapeutic efficacy by causing the liposomes to release the encapsulated API or cargo based on a stimulus response. LNPs used to deliver genes are primarily synthesized using cationic, or positively-charged, lipids that associate with anionic, or negatively-charged, nucleic acids. Another common lipid component is the so-called PEGylated phospholipid-. These usually include neutral phospholipid molecules belonging to the phosphatidylcholine (PC) class and sterols such as cholesterol. ), and has accumulated a large number of data models and rich research experience in the construction and optimization of nanocarriers for gene vaccines and protein drugs. Second, they serve to help solubilize highly lipophilic drug molecules or modulate the pharmacokinetics and biodistribution of the APIthereby helping to minimize side effects and enhance the product safety profile. Second, they serve to help solubilize highly lipophilic drug molecules or modulate the pharmacokinetics and biodistribution of the APIthereby helping to minimize side effects and enhance the product safety profile. Because so much of the growing field of personalized medicine is focused on genetic therapies, LNPs have become particularly useful as a drug delivery platform. We specialize in the development and manufacture of lipid-based, parenteral drug products, and our team has more than 20 years' experience working with a wide range of preclinical, clinical and commercial contract manufacturing customers. LNP drugs have cropped up across the pharmaceutical industry as therapies designed to deliver anti-cancer agents, antibiotics, gene medicines, anesthetics and anti-inflammatory drugs. References: Other lipid-based components can also be added to modulate the delivery efficiency and location release of the genetic cargo. Liposomes possess a unique vesicular structure. While this is advantageous and enables these lipid-based particles to assume useful applications in hundreds of different settings, the number of potential variations makes rigorous manufacturing control imperative. As such, any cargo of interest can be encapsulated within liposomes in either the aqueous compartment (if it is water-soluble/hydrophilic) or within the lipid bilayer (if fat-soluble/lipophilic). Their self-closed structures can encapsulate multiple drugs at once, protecting enclosed cargo from hydrolysis and breakdown. Journal of materials chemistry B, Materials for biology and medicine. Patient or antigen-specific drugs dont leave much time from the moment the oligonucleotide is sequenced to the time the product needs to be formulated at our site. doi:10.1039/C3TB21238F. LNP drugs have cropped up across the pharmaceutical industry as therapies designed to deliver anti-cancer agents, antibiotics, gene medicines, anesthetics and anti-inflammatory drugs.3, In clinical applications, liposomal drugs have been proven to be most useful for their ability to passively accumulate at sites of increased vasculature permeability, when their average diameter is in the ultrafilterable range (<200 nm in diameter), and for their ability to reduce the side effects of the encapsulated drugs relative to free drugs. Their amphiphilic structure allows encapsulation of both hydrophilic and hydrophobic active pharmaceutical ingredients (APIs). Amphotericin B, the active ingredient in Abelcet, is held within a lipid complex and selectively fuses with fungal membranes to target disease cells. Advanced Drug Delivery Reviews. Current Nanomedicines for the Treatment of Cancer In clinical applications, liposomal drugs have been proven to be most useful for their ability to, accumulate at sites of increased vasculature permeability, when their average diameter is in the ultrafilterable range (, 200 nm in diameter), and for their ability to reduce the side effects of the encapsulated drugs relative to free drugs. 2018 Jan; [. Typically, liposomes are manufactured as sterile injectables for delivery to the bloodstream, and release of the drug takes place when lipid envelopes break downwhich can happen in extracellular or intracellular environments. For a long time, the most effective way to deliver gene-based therapeutics to human cells was to use a virus that had been modified to carry medicinal cargo rather than harmful, self-replicating genes. They can both deliver their payload very precisely through treating their surface with proteins allowing highly specific binding to a target cell type. Advantages and Disadvantages of PEGylated Liposomes in Drug Delivery System While this is advantageous and enables these lipid-based particles to assume useful applications in hundreds of different settings, the number of potential variations makes rigorous manufacturing control imperative.2. Non-viral gene delivery, however, has become popular over the last. The preparation of liposomes with entrapped solutes was first demonstrated in a published paper by Prof. A.D. Bangham of the United Kingdom. Therefore, it is also called "drug missile"; lipid nanoparticles have a continuous bilayer that would qualify them as lipid vesicles or liposomes. Some lipid nanoparticles are micellar-like structures, encapsulating drug molecules in a non-aqueous core. For example, amphotericin B liposomes can reduce cardiotoxicity. One of the primary drivers of this movement has been the development of lipid and polymer-based carriers, of which LNPs are the most popular. Since their inception, liposomes have been explored as carriers for delivering drugs and pharmaceuticals. These vesicles are composed of a lipid bilayer that is primarily composed of amphipathic phospholipid enclosing an interior aqueous space. LNPs also provide mechanical stability, controlled morphology and narrow size distribution. Liposomal drug delivery systems: From concept to clinical applications. ), and has accumulated a large number of data models and rich research experience in the construction and optimization of nanocarriers for gene vaccines and protein drugs. Liposomes and LNPs have application as delivery vehicles for each of these categories of drug products, making them an indispensable asset in this new field of pharmaceutical development. Unfortunately, delivery of free, unencapsulated RNA into human cells is difficult, as they are large, unstable in serum and prone to nuclease degradation. Wan C, Allen TM, Cullis PR. Because so much of the growing field of personalized medicine is focused on genetic therapies, LNPs have become particularly useful as a drug delivery platform. In contrast, traditional manufacturing batches for mainstream pharmaceuticals often produce thousands of liters of drug product at scale. Lipid nanoparticle delivery systems for siRNA-based therapeutics. Since their inception, liposomes have been explored as carriers for delivering drugs and pharmaceuticals. This can result in loss of the encapsulated drug or unfavorable mixing of different vesicles cargo. This may result in loss of encapsulated drug or unfavorable mixing of different vesicles cargo. years due to enhanced safety profiles, lower rates of adverse immunogenic reactions and ease of manufacturing. Reduce drug toxicity: Liposome phospholipid bimolecular membrane is similar to mammalian cell membrane, which reduces the body's immune response and is not easy to cause immune response such as allergies. In addition, various physicochemical properties of liposomesincluding their size, charge, and surface functional ligandscan be altered, resulting in functionalities favoring specific drug delivery tasks. [3] Theresa M. Allen, Pieter R. Cullis. One of the most successful variations of these hybrid nanoparticles incorporates PLA or PLGA polymers within a lipid monolayer. First of all, the preparation technology brings some difficulties to commercial production; in addition, for some water-soluble drugs, the encapsulation rate is low, and the drugs are easy to leak from the liposomes; poor stability is also a problem that needs to be solved in the commercialization of liposomes, and the current lyophilization method may be an effective way to extend the storage period of liposomes. US News: Health Care. More recently the liposomes analogous cousin, the lipid nanoparticle, has gained prominence because of its ability to deliver therapeutic payloads, including DNA and mRNA for vaccines. This has resulted in an overall increase in therapeutic index, which measures efficacy over toxicity., This is extremely applicable for diseases like cancer. 2014;103(1):29-52. doi:10.1002/jps.23773. 2014;103(1):29-52. doi:10.1002/jps.23773. Exelead has been manufacturing lipid-based drugs since the early 1990s, starting withAbelcet. These two core biopolymers are particularly useful in drug delivery because they facilitate controlled drug release. To help improve transportation efficiency and allow more circulation time for the cargo molecules to reach the expected disease site, PEG is added to shield these nanoparticles by preventing blood plasma proteins from absorbing into the liposome surface, increasing bloodstream circulation lifetime. Improve stability: Drugs stored for a long time are prone to deterioration, but under the protection of the liposome molecular layer, the possibility of oxidative degradation of the drug is greatly reduced, thereby prolonging the drug effect; In recent years, liposomes have attracted significant attention as a trusted class of drug delivery vehicles. The nanoparticle formulation shown above is a lipid-polymer hybrid with a hydrophobic PLGA core and a hydrophilic lipid-PEG shell. As such, any cargo of interest can be encapsulated within liposomes in either the aqueous compartment (if it is water-soluble/hydrophilic) or within the lipid bilayer (if fat-soluble/lipophilic). One way that drug manufacturers have learned to overcome this problem is by covering the exterior of liposomes with polymers such as PEG. Lipid nanoparticles are liposome-like structure, which is especially suitable for encapsulating various nucleic acids (RNA and DNA). Liposomes and Lipid Nanoparticles as Delivery Vehicles for Personalized Medicine, At Exelead, we approach contract manufacturing with focused expertise on, We specialize in the development and manufacture of lipid-based, parenteral drug products, and our team has more than 20 years' experience working with a wide range of, Liposomes are specialized delivery vehicles. Advanced Drug Delivery Reviews. As such, any cargo of interest can be encapsulated within liposomes in either the aqueous compartment (if it is water-soluble/hydrophilic) or within the lipid bilayer (if fat-soluble/lipophilic). As personalized medicine has become a prominent focus in drug development, many companies in the pharmaceutical manufacturing industry have. Gao W, Hu C-MJ, Fang RH, Zhang L. Liposome-like Nanostructures for Drug Delivery. Liposomes possess a unique vesicular structure. Journal of pharmaceutical sciences. [2] Kraft JC, Freeling JP, Wang Z, Ho RJY. It is certain that liposome-like nanocarriers will play a larger role for drug delivery in the foreseeable future., - Liposome-like nanostructures for drug delivery|Gao W, Hu C-MJ, Fang RH, Zhang L1. Some of the primary lipids used to make liposomes are phospholipids and sphingolipids. In theory, segments of siRNA can be designed to silence any gene, which is an exciting concept for both doctors and researchers. particular genetic mutation a patient has developed allows doctors to employ more specific and precise treatments. Liposomes are specialized delivery vehicles that serve multiple roles in enhancing the capabilities of active pharmaceutical ingredients (APIs). As personalized medicine has become a prominent focus in drug development, many companies in the pharmaceutical manufacturing industry have adapted their pipelines to accommodate smaller batches slated for small groups of patients in addition to traditional, large-scale drug production. Any oligonucleotide could theoretically be encapsulated within a liposome or LNP, but siRNA. Non-viral gene delivery, however, has become popular over the last 20 years due to enhanced safety profiles, lower rates of adverse immunogenic reactions and ease of manufacturing. In contrast with traditional, big-pharma approaches to treatment of disease, personalized medicine takes into account individual differences in lifestyle, environment, and biologyincluding a patients genetics. LNPs are composed primarily of cationic lipids(see gene therapy below) along with other lipid ingredients. This has resulted in an overall increase in therapeutic index, which measures efficacy over toxicity., - Liposomal drug delivery systems: from concept to clinical applications | Allen TM, Cullis PR 3. (, https://www.genengnews.com/insights/nonviral-crispr-delivery-vehicles-lay-the-smart-siege/, https://www.exeleadbiopharma.com/news/liposomes-and-lipid-nanoparticles-as-delivery-vehicles-for-personalized-medicine, https://www.sciencedirect.com/science/article/pii/B9780323428682000127, https://pubs.acs.org/doi/10.1021/acsami.0c16380, https://www.medgadget.com/2019/07/lipid-nanoparticles-deliver-crispr-cas9-into-organs-with-high-efficiency.html. This method is still occasionally used today, and is referred to as viral gene delivery. Targeting: Liposomes can selectively enter certain tissues or organs of the human body, such as liver and spleen. Some LNPs assume a micelle-like structure, encapsulating drug molecules in a non-aqueous core. Traditional liposomes include one or more rings of lipid bilayer surrounding an aqueous pocket, but not all LNPs have a contiguous bilayer that would qualify them as lipid vesicles or liposomes. 2014 Feb;4(1):74-83. doi: 10.1007/s13346-013-0161-z. To aid in delivery efficiency and to allow more circulation time for cargo molecules to reach intended diseases sites, PEG is added to shield these nanoparticles by preventing blood plasma proteins from absorbing into the liposome surface, increasing bloodstream circulation lifetime. In the 1990s a need was identified for alternate approaches for nanoparticles based on lipid components other than phospholipids. Exelead develops and manufactures LNPs to encapsulate different types of genetic payloads including, PEGylated phospholipids are used in many lipid-based drug carriers primarily because they offer what is known as a stealth effect to the drug product as it circulates within the body. The concentration of liposome drugs in the liver is 200 to 700 times that of ordinary drugs. In contrast, traditional manufacturing batches for mainstream pharmaceuticals often produce thousands of liters of drug product at scale. Proprietary lipid technology is used for each. Traditional liposomes include one or more lipid bilayer rings surrounding an aqueous pocket, but not alllipid nanoparticles have a continuous bilayer that would qualify them as lipid vesicles or liposomes. Conventional liposomes, especially liposomes smaller than 200 nm in size, may themselves be unstable and tend to fuse with each other to reduce surface tension. , protecting the siRNA segments until they reach their intended destination and facilitating their delivery into target cells. Another common lipid ingredient is what is known as a PEGylated phospholipida polyethylene glycol (PEG) polymer covalently attached to the head-group of a phospholipid. Alternatively, thermosensitive or photosensitive components are sometimes included to enable breakdown and structure modulation due to changes in temperature or reaction to light of certain wavelengths. Both are lipid nanoformulations and excellent drug delivery tools that can transport targeted cargo within the protective outer layer of lipids. [NCBI] BothRNA vaccinesconsist of Messenger RNA, or mRNA, encased in a bubble of oily molecules called lipids. Emerging Research and Clinical Development Trends of Liposome and Lipid Nanoparticle Drug Delivery Systems. has been focusing on the development of a full range of medical applications and technologies for nanocarrier systems (including various types of nanoparticles, liposomes, micelles, etc. The second benefit of PEGylation is that it improves the stability of liposome-like nanostructures. Stealth Liposomes (PEGylated Liposomes) as Drug Carrier System for Drug Delivery Journal of pharmaceutical sciences. One way drug manufacturers have learned to overcome this problem is by covering the exterior of liposomes with polymers like PEG. polymers within a lipid monolayer. Personalized drug products are often manufactured in small batch sizes for single patients or small populations and can frequently result in less than one liter of product. Recently, PEGylated lipids attracted more and more attention as a PEGylated lipid is used asan excipient in both theModerna vaccineand thePfizerBioNTech COVID-19 vaccine. One way drug manufacturers have learned to overcome this problem is by covering the exterior of liposomes with polymers like PEG. The human immune system is driven to protect the body from any foreign object, and medicinal nanoparticles are no exception. These stealth-equipped nanoparticles have led to a new generation of liposome preparations and a variety of clinically approved products. Stealth Liposomes (PEGylated Liposomes) as Drug Carrier System for Drug Delivery, Current Nanomedicines for the Treatment of Cancer, Advantages and Disadvantages of PEGylated Liposomes in Drug Delivery System, Lipid Nanoparticles for Drug and Vaccine Delivery, Cyclic Peptide Therapeutics: R&D Progress, Oligonucleotide Drugs: Current Status and Challenges. The second benefit of PEGylation is a boost in stability for liposome-like nanostructures. Journal of materials chemistry B, Materials for biology and medicine. LNPs used to deliver genes are primarily synthesized using cationic, or positively-charged, lipids that associate with anionic, or negatively-charged, nucleic acids. In theory, segments of siRNA can be designed to silence any gene, which is an exciting concept for both doctors and researchers. Unfortunately, delivery of free, unencapsulated RNA into human cells is difficult, as they are large, unstable in serum and prone to nuclease degradation.5, While researchers have made attempts to stabilize siRNA in serum by adding phosphorothioate linkages, high doses are required to effectively silence genes in humans. These ligands attach to cell receptors that are over-expressed in certain diseased cells, allowing entry of the drug through the cell membrane. 2013 Jan;65(1):3648, Related Articles: polymer covalently attached to the phospholipid head group. With theadventof personalized genetic therapies, doctors and scientists can effectively tailor an active pharmaceutical ingredientoften RNA or DNAto match the specific disease profile of a particular patient or small group of patients. Any oligonucleotide could theoretically be encapsulated within a liposome or LNP, but siRNA are currently the most common cargo in these types of drug products. Therefore, it is also called "drug missile"; These two categories of lipids are unique in terms of a head group that is water-loving/hydrophilic and a tail group that is water-hating/lipophilic. Exelead has been manufacturing lipid-based drugs since the early 1990s, starting with, . often manufactured in small batch sizes for single patients or small populations and can frequently result in less than one liter of product. Lipid Nanoparticles represent a relatively new colloidal drug delivery system. While personalized medicine has the potential to treat almost any disease, current research has primarily focused on 1) immunotherap. PEGylated liposomes and LNPs are currently the new paradigm for most cancer therapeutics. These usually include neutral phospholipid molecules belonging to the phosphatidylcholine (PC) class and sterols such as cholesterol. [. Currently, a number of liposome formulations are in clinical use to deliver anticancer, anti-inflammatory, antibiotic, antifungal, anesthetic, and other drugs and gene therapies, while others await clinical trial outcomes (for updated information, please visit the website www.clinicaltrials.org). Due to their amphiphilic nature, these molecules spontaneously self-assemble to form liposomes and other unique 3D structures when added to aqueous solutions. Additionally, targeting proteins and surface functional ligands on the outer shell of the lipid bilayer can add novel functionalityenabling targeted entry of liposomes into cells, either via antibodies or receptor-targeted ligands. These vesicles are composed of a lipid bilayer that forms in the shape of a hollow sphere encompassing an aqueous phase. Vogenberg FR, Isaacson Barash C, Pursel M. Personalized Medicine: Part 1: Evolution and Development into Theranostics. These vesicles are composed of a lipid bilayer that forms in the shape of a hollow sphere encompassing an aqueous phase. At Exelead, extensive efforts have been made to accommodate these small-batch therapeutics, which often require expensive API and quick turnaround time. Since liposomes were first proposed as a drug delivery system in the late 1960s, variations in structure and functionality have emerged, providing valuable advancements in terms of disease targeting. Exelead develops and manufactures LNPs to encapsulate different types of genetic payloads including siRNA, mRNA and saRNA. However, in applications, lipid nanoparticles can take many forms. Liposome-like drug carriers can come in many different varieties, exhibiting a wide range of biochemical and biophysical properties. Formulations geared for release in intracellular environments can include pH-sensitive lipids that change the liposomal structure or degrade within acidic compartments, enabling the release of the encapsulated drug. LNPs have provided a solution to this problem by providing flexible and easy means of encapsulation, protecting the siRNA segments until they reach their intended destination and facilitating their delivery into target cells.5, LNPs containing ionizable cationic lipids have a number of features necessary for the systemic delivery of polynucleic acids, including small sizes, serum stability, low surface zeta potentials at physiological pH, and cationic charge at acidic pH values (e.g., in endosomes). This method is still occasionally used today, and is referred to as viral gene delivery. Liposomes possess a unique vesicular structure. These stealth-equipped nanoparticles have resulted in a new generation of liposomal formulations and multiple clinically-approved products. Drug Deliv Transl Res. Since liposomes were first proposed as a drug delivery system in the late 1960s, variations in structure and functionality have emerged, providing valuable advancements in terms of disease targeting. It drives the human immune system to protect the human body from any foreign bodies, and medicinal nanoparticles are no exception. [, Theresa M. Allen, Pieter R. Cullis. a stabilizing molecule of polyethylene glycol. that serve multiple roles in enhancing the capabilities of active pharmaceutical ingredients (APIs). To help improve transportation efficiency and allow more circulation time for the cargo molecules to reach the expected disease site, PEG is added to shield these nanoparticles by preventing blood plasma proteins from absorbing into the liposome surface, increasing bloodstream circulation lifetime. Other lipid-based. All Rights Reserved Copyright 2013-2021. To aid in delivery efficiency and to allow more circulation time for cargo molecules to reach intended diseases sites, PEG is added to shield these nanoparticles by preventing blood plasma proteins from absorbing into the liposome surface, increasing bloodstream circulation lifetime.1. These two core biopolymers are particularly useful in drug delivery because they facilitate controlled drug release.1. Liposomes and lipid nanoparticles (LNPs) are similar by design, but slightly different in composition and function. 2013;1(48):10.1039/C3TB21238F. Inorganic materials, organic materials and hydrogels have. Liposomes and lipid nanoparticles are similar in design, but slightly different in composition and function. PEGylated liposomes and lipid nanoparticles are currently thenew paradigmsfor most cancertherapeutics. 2013;1(48):10.1039/C3TB21238F. PEGylated liposomes and LNPs are currently the, For a long time, the most effective way to deliver gene-based therapeutics to human cells was to use a virus that had been modified to carry medicinal cargo rather than harmful, self-replicating genes. This approach to hyper-specific disease targeting increases efficacy and decreases unwanted side effects for groups of similar patients. PEGylated phospholipids are used in many lipid-based drug carriers primarily because they offer what is known as a stealth effect to the drug product as it circulates within the body. Liposomal drug delivery systems: From concept to clinical applications. Even within a single type of cancer, tumor types differ from one patient to another, and understanding the particular genetic mutation a patient has developed allows doctors to employ more specific and precise treatments.4. The second benefit of PEGylation is a boost in stability for liposome-like nanostructures. Conventional liposomes, particularly those smaller. The preparation of liposomes with entrapped solutes was first demonstrated in a published paper by Prof. A.D. Bangham of the United Kingdom. Exelead is taking on such challenges, including the development of nanogels that incorporate an array of biologics and small molecules. ; Lipid nanoparticlesare mainly composed of cationic lipids and other lipid ingredients. It drives the human immune system to protect the human body from any foreign bodies, and medicinal nanoparticles are no exception. While personalized medicine has the potential to treat almost any disease, current research has primarily focused on 1) immunotherapies, 2) conventional therapies augmented via pharmacogenomics and 3) biomarker-related cancer treatments. [1] Gao W, Hu C-MJ, Fang RH, Zhang L. Liposome-like Nanostructures for Drug Delivery. Liposomes are the most studied drug carriers due to the biocompatibility and biodegradability that they present. 2010;35(10):560-576. These typically include neutral phospholipid molecules belonging to the phosphatidylcholine (PC) class and sterols, such as cholesterol. LNPs are liposome-like structures especially geared towards encapsulating a broad variety of nucleic acids (RNA and DNA); and as such, they are the most popular non-viral gene delivery system. Personalized medicine requires a unique approach, and each batch must be manufactured under stringent cGMP conditions. This release is either driven by conventional breakdown of the liposomes, or it is driven by active stimuli and environmental cues, including thermal energy, pH gradient and shear stress. Further, by taking advantage of endogenous targeting processes due to association with ApoE following administration, highly efficient uptake into hepatocytes can be achieved following i.v. Biochempeghas been focusing on the development of a full range of medical applications and technologies for nanocarrier systems (including various types of nanoparticles, liposomes, micelles, etc. Lipid Nanoparticles for Drug and Vaccine Delivery. PEGylated phospholipids are used in many lipid-based drug carriers primarily because they offer what is known as a stealth effect to the drug product as it circulates within the body. These short-term forecasts, sometimes only six weeks, present challenges that we have been able to overcome by refining our existing systems and incorporating innovative formulation techniques. Since their discovery in 1965, by Alec D. Bangham, liposomes have been recognised as the drug delivery vehicle of choice. One of the primary drivers of this movement has been the development of lipid and polymer-based carriers, of which LNPs are the most popular. In both vaccines, the bubbles are coated with a stabilizing molecule of polyethylene glycol. The concentration of liposome drugs in the liver is 200 to 700 times that of ordinary drugs. [NCBI] Pharmacy and Therapeutics. any foreign object, and medicinal nanoparticles are no exception. Currently, a number of liposome formulations are in clinical use to deliver anticancer, anti-inflammatory, antibiotic, antifungal, anesthetic, and other drugs and gene therapies, while others await clinical trial outcomes (for updated information, please visit the website. This release is either driven by conventional breakdown of the liposomes, or it is driven by active stimuli and environmental cues, including thermal energy, pH gradient and shear stress.1. 200 nm in size, can be unstable on their own and tend to fuse with each other to reduce surface tension. First, they can shield a drug from detection by the bodys immune system, mimicking biological membranes and giving the drug more time to reach its intended destination.
Sitemap 9