Formulation of biotechnological drugs

Formulation of biotechnological drugs

Polymers

Polymer: special macromolecule composed by repeated units covalently linked to each other to form compounds of high MW, represented by n which is a quite high number. Polymers are obtained through polymerization of monomers. [A] → [CH₂-CH₂-O]_n

Oligomers: very small polymers, having a low MW and a number of repeating units typically less than 10.

Types of polymers

• Homopolymers: based on a single monomer -A-A-A-A-A-A-A-
• Copolymers: based on different monomers.
• Alternating copolymers: regular alternating A and B units -A-B-A-B-A-B-A-B-A-.
• Statistical copolymers: copolymers in which the sequence of monomer residues follows a statistical rule. If the probability of finding a given type monomer residue at a particular point in the chain is equal to the mole fraction of that monomer residue in the chain, then the polymer may be referred to as a random copolymer -A-B-B-B-B-A-A-B-A-B-B-A-.
• Block copolymers: comprise two or more homopolymer subunits -A-A-A-B-B-B-B-A-A-A-B-B-B-B-A-A-A-.
• Graft copolymers: formed by polymer chains grafted on other polymer chains.

Polymers structures

• Dendrimers: large, synthetically produced polymers in which the atoms are arranged in many branches and subbranches radiating out from a central core. Dendrimers are being investigated for possible uses in nanotechnology, gene therapy, and other fields.
  • Backfolding: it may happen that not all the terminal groups are exposed to the surface of the dendrimer. This affects the complete branching of all terminal groups and also can affect the drug loading.
• Dendronized polymers.
• Graft polymers.
• Block copolymers.
• Multivalent polymers.
• Branched polymers.
• Stars.

Crosslinked polymers: formed from polymer chains joined together with bridges of various nature and length; such polymers develop in space assuming a 3D structure.

Polymers in solution are free to move, depending on the interactions between chains there can be more or less viscosity. But if the structure is cross-linked they can’t, creating a hydrogel that keeps water inside.

Main physicochemical and mechanical properties of a polymer

• MW: it determines most of the characteristics. Example: a short polymer would be soluble, freely to move, ideal for viscous solutions but not hydrogels. Also, it determines if a polymer can be cleared or not by the kidney.
• Volume: the hydrodynamic volume characteristic for each polymer. It's really different between a hydrophilic polymer and a more hydrophobic one, because of the presence/absence of water. A hydrophilic one is a better choice to protect a protein because it covers better its surface.
• Hydrophilicity/hydrophobicity balance.
• Solubility: in water.
• Degradability: if the bonds between monomers are degradable in vivo, so a polymer with a higher MW can be used. If it’s not biodegradable, it must be cleared by the body, otherwise, it will be accumulated. Any substance smaller than albumin (60 kDa, 10 nm long) can be cleared by the kidney, but with polymers, the volume must be considered, so the size not the MW.
• Hardness
• Fragility: how elastic is the polymer without breaking.
• Charge: some groups aren’t charged at physiological pH, but if pH is raised, they get a negative charge and become soluble.
• Plasticity
• Crystallinity
• Viscosity
• Chemical reactivity.

These properties depend on: chemical nature of monomer(s), relative proportions of the monomers, molecular weight, conformational structure, degree of crosslinking, covalent bonds between monomers, charges, degree of polymerization.

Classification

The polymers can be classified in several ways, but a definition does not exclude the other.

Origin

• Natural polymers: polysaccharides, proteins, nucleic acids.
• Synthetic polymers: plastics, resins, gums, gels. Higher polydispersity, because there’s more control.

Thermic behavior

• Thermoplastic: T increases, the viscosity increases.
• Thermosetting: usually there are crosslinked bonds, so the increase of T makes them more rigid.

Commonly the polymers are present in an amorphous state which in turn can be either glassy or rubbery material depending on the temperature. The temperature of transition from one state and the other is defined glass transition temperature (Tg), this is typical of each polymer and can vary over a wide range.

By freezing a rubbery state, it passes to a glassy state and can be broken since it’s not elastic anymore, so polymers can be present as liquid or solid depending on their characteristics and temperature. The Tg allows the prediction of some characteristics of the polymer.

• If an amorphous polymer has a Tg < room temperature will have a rubbery behavior and can plastically and elastically deform. The polymer is defined elastomer.
• If a polymer has a Tg > room temperature, it will be hard and glassy and is defined thermoplastic. A thermoplastic polymer can achieve the rubbery state by heating, raising the T above its Tg value. Therefore the elastomers have a Tg lower than the thermoplastic.

Typically the polymers in the liquid state are viscous fluids. A complete crystal structure is almost never reached by polymers but usually can have semi-crystalline structures in which only certain areas assume an ordered structure.

Molecular weight and polydispersity

The molecular weight of a polymer determines many important properties of the polymer itself and is expressed in different ways depending on the technique used for its determination:

• Numeral average molecular weight: Mn = ∑ni·Mi/∑ni
• Weight average molecular weight: Mw = ∑ni·Mi /∑ni·Mi

Where ni=n1, n2, n3 ... are the molecules of polymer with a mass Mi=M1, M2, M3 ...

A polymer is made of a mixture of chains of different length with a different weight. It’s impossible that all chains have the same MW. So the MW of the polymers is an average value of all MWs. The MW can be represented as a Gaussian with a small amount of high-MW chains and a small amount of low-MW chains, the average in the middle characterized most of the chains.

The polymers, generally, are never composed of macromolecules having all the same molecular weight. They are mixtures of similar macromolecules with different molecular weights. This variability originates from the polymerization reaction, because its typical property of being a chain reaction means that it begins, proceeds and ends in a more or less random way, thus generating polymers of variable length. The minimum difference between two macromolecules corresponds to the molecular weight of the monomer.

The polydispersity is the ratio between Mw and Mn and it gives an idea of the purity of a polymer. Polymers with low polydispersity values will have well-defined characteristics because the molecules that compose the polymer have more or less the same molecular weight.

• Polymers with a low polydispersity → very narrow Gaussian distribution (polydispersity ≈ 1).
• Polymers with a high polydispersity → very broad Gaussian distribution (high polydispersity values).
• Proteins have a polydispersity = 1, since all the chains have the same MW, while for polysaccharides it is 2-3.

Examples:

• A theoretical polymer mixture composed of one molecule of 900 Da, 2 molecules of 1000 Da and one molecule of 1100 Da: Mn = 1000 Da; Mw = 1005 Da; Polydispersity = 1.005
• Another polymer mixture composed of two molecules of 900 Da, one of 1000 Da, one of 1100 Da: Mn = 975 Da; Mw = 982 Da; Polydispersity = 1.007

To improve the polydispersity of a polymer it is possible to perform one or more of the following purifications:

• Gel filtration.
• Solvent precipitation.
• Ultrafiltration.

Role of functional groups

The chemical groups present in the polymer play a key role in defining its physicochemical properties. The solubility in water is related to the presence of hydrophilic groups, while the solubility in organic solvents is given by alkyl residues or other hydrophobic groups. The simultaneous presence of hydrophilic and hydrophobic groups allows the solubility both in water and in organic solvents.

The simple quali/quantitatively modification of the monomers can produce polymers with the most different physicochemical characteristics. Furthermore, it’s possible to add modifying agents (like crosslinkers) or to use mixtures of different polymers to further calibrate the characteristics of a polymer. All this has allowed the extensive development and applicability of polymers in almost all sectors.

From the pharmaceutical point of view, the polymers represent basis materials for the preparation of many traditional pharmaceutical forms and for the development of drug delivery systems for modified and controlled drug release. They have both basic and very advanced uses. Uses of polymeric excipients in the classic pharmaceutical technology: fillers, binders, disintegrants, coating materials, rheology modifiers, surfactants, etc.

Polymers with particular characteristics:

• With charged chemical groups: solubility as a function of the pH, it will be greater when the groups are charged (protonated/deprotonated) and lower when the pH value is close to the isoelectric point.
• With pendant hydrophobic groups along the backbone: solubility as a function of the temperature. At low T, they are soluble by forming rigid structures in which the water is coordinated around the pendant groups. The increase in T causes an increase of the energy of the system and the water molecules are detached, thus leading to the collapse of the hydrophobic groups and the precipitation of the polymer. The temperature at which this transition occurs is called low critical solution temperature (LCST).

Biodegradable polymers

The biodegradability of a polymer is determined by the type of bond that links together the monomers: hydrolysable bonds allow the progressive degradation of the polymer chain up to the formation of the base units. To this class belong: polyesters, polyorthoesters, polycarbonates, polyphosphazenes, poliglutamic acid, etc. Toxicity can derive from impurities in a polymer.

Polymers of small dimensions (up to 32 Aj) have usually predominantly renal clearance, which depends on various structural characteristics, but in general, increases with the decrease of the MW. Polymers with high MW are not eliminated from the body and are progressively accumulated in the liver with toxic effects ("macromolecular syndrome"). Therefore it’s important to use biodegradable polymers when they are not easily eliminated from the body.

Biocompatibility is a very important parameter, but it’s difficult to predict and there are no general rules. Among the most biocompatible materials, there are very hydrophobic materials, such as silicones, or very hydrophilic polymers such as polyvinyl alcohol. Even the bioadhesion, the ability to adhere to biological surfaces, is found in polymers with very different chemical-physical properties.

Classes of polymers

• Silicones: Not organic, backbone has no C atoms. Many applications: in the medical field, they are used as heart valves, pacemakers components, catheters, etc; in the drug delivery field, they are employed for extended-release dosage forms. Their characteristics are determined by the length of the chain and the substituent R. They are elastic thanks to the flexibility of the O-Si-O bonds. Common characteristics: physical stability, chemical resistance, flexibility, hydrophobicity, biocompatibility. They can be linear or cross-linked.
• Polyesters of α-hydroxyacids: Polylactic acid (PLA) and Polyglycolic acid (PGA). Used for nanoparticles, biodegradable, easily hydrolysable in vivo. PLA is more hydrophobic than PGA, so it tends to hydrolyze slower.
• Polyacrylates: They belong to the family of vinyl polymers and they are obtained by radical polymerization of vinyl monomers. Even in this case, the group R determines a wide variability in the characteristics; also the possibility of synthesizing copolymers allows further expansion of the properties of this family of polymers. They are used in soft contact lenses (polyacrylamide), as carriers of soluble drug and as matrices for drug delivery.
• Polyvinyl alcohol (PVA): Very hydrophilic, with a solubility as a function of the MW. Widely used in the formation of hydrogels for drug delivery. Used to prepare hydrogel in the lab: from a solution, put the drug/protein and then freeze and thaw more times. Creation of crystalline areas that represents contact points.
• Polyethylene glycol (PEG): Leading polymer used for covalent attachment to a surface. Soluble in aqueous solution and in many organic solvents, not toxic, not immunogenic and not antigenic. It’s not biodegradable, but it’s easily cleared from the body by renal or hepatic excretion on the basis of its MW. Approved by the FDA for human use.

Liposomes

A liposome defines a closed vesicular system commonly formed of phospholipids, arranged in highly ordered double layers called bilayers. The bilayers separate two aqueous compartments, one internal and one external, in which the liposome is suspended. In addition to phospholipids, the bilayers can be composed of other lipid substances.

Phospholipids

They are amphiphilic substances, consisting of a polar head, a phosphoric residue eventually substituted with other organic groups, and two apolar aliphatic tails, joined by a molecule of glycerol. When phospholipids are dispersed in an excess of water, they orient themselves forming a double molecular layer (similar to biological membranes) in which the hydrophilic heads are oriented towards the aqueous medium while the hydrophobic tails remain in close contact with each other. The bilayers close themselves to form concentric vesicles which are called liposomes. Phospholipids are the main constituents of liposomal systems and biological membranes.

The closing of the bilayers in forming the liposomes allows trap of molecules of various kinds (liposoluble and hydrosoluble), therefore the liposome systems can act as a "carrier". The hydrophobic tails form the hydrophobic compartment, which is separated from the surrounding polar solvent.

The phosphoric group of the hydrophilic head in a phospholipid is esterified (with the exception of phosphatidic acid) with the -OH of strongly hydrophilic molecules such as variously substituted amines, carbohydrates or amino acids. The hydrophilic head can have a net charge (positive or negative), or can be neutral. A net charge equal to zero is important, otherwise, the heads will repel each other and can’t form the bilayer. This part of the molecule of the phospholipid takes the name of the polar head, while the part of the molecule formed by the two aliphatic chains takes the name of the hydrophobic zone of the phospholipid. The tail can be short or long and have an effect on the liposome: by prolonging the tail, the bilayer would be more thick. Also, hydrophobic interactions keep the phospholipids together, so increasing the length of the tails, the interactions and so the liposome would be more stable.

To get inside, the drug must cross the bilayer, so with a hydrophilic one, it would be more difficult, while a hydrophobic drug will be stuck in the bilayer.

A liposome has a water compartment inside, micelle doesn’t, so can’t carry hydrophilic drugs, just hydrophobic. The shape of the amphiphilic molecule determines the formations of micelles or liposomes. A conic-shaped molecule like a lipid, with only one tail and the head bigger than the space occupied by the hydrophobic moiety, is ideal for micelles. A cylinder-shaped one like phospholipids is used for liposomes since they aggregate forming the bilayers.

Most common phospholipids: phosphatidylcholine, phosphatidylethanolamine (+), phosphatidylglycerol, phosphatidylserine (-), phosphatidic acid, and phosphatidylinositol. Among the various types of phospholipids, lecithin is the most important class of compounds both from the biological point of view and from the point of view of pharmaceutical application. They are the main constituents of biological membranes. They are the most used substances in cosmetic and pharmaceutical preparations.

Other constituents of liposomes

The glycolipids are molecules of lipids that do not present the phosphoric group. The main classes are the gangliosides, sulfatides, and cerebrosides. Sometimes they are used as constituents of liposomes, they have the ability to give vesicular systems with characteristics of long circulation in the human body.

Cholesterol is one of the most important constituents of biological membranes and often also of liposomes. It has the characteristic to modulate the physicochemical properties and the morphological-functional characteristics of bio-membranes. It specifically regulates the fluidity of the bilayers. Cholesterol is very hydrophobic so it interacts with the tails, only the -OH is exposed on the membrane surface. The cholesterol balances the situation.

• T<Tm: cholesterol reduces the order of the aliphatic chain of fatty acids and it obstacles the crystallization, since it interferes with the interaction between phospholipids chains. So the fluidity is increased.
• T>Tm: cholesterol increases the order of fatty acids chains, so there’s less fluidity.

Liposome as drug delivery system

Reasons to use them:

• Solubilization: improve the solubilization of a hydrophobic drug.
• Protection: encapsulated drugs are inaccessible to metabolizing enzymes, conversely, body compartments are not directly exposed to the full dose of the drug.
• Duration of action: can prolong drug action by slowly releasing.
• Directing potential: targeting options change the distribution through the body.
• Internalization: liposomes are endocytosed or phagocytosed by cells, opening up opportunities to use "liposome-dependent drugs". Lipid-based structures are also able to bring plasmid material into the cell through the same mechanism.
• Amplification: can be used as adjuvants in vaccine formulations.