Biomedical Engineering - Nanomedicine - Principles and Applications

Completed notes of the course

Complete course

1. Basic Principles To figure out what nanomedicine means there is simply to remind the words from which such term comes from: a combination of “nanotechnology” and “medicine”. So , the feeling is that any sort of nanotechnology applied for medicine. For example, let’s think about drug delivery , o r hydroge ls for tissue engineering : whatever involves the use of nanotechnology for curing diseases is roughly what nanomedicine is about. :owever, we find out soon that an exact definition of nanomedicine does not exist . All of them are similar, but not the same. For example : “The science and technology of diagnosing, treating, and preventing disease and traumatic injury, of relieving pain, and of preserving and improving human health, using molecular tools and molecular knowledge of the human body ”. “An offshoot of nanotechnology, which refers to highly specific medical interventions at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve ”. The main problem is to define what nanotechnology means or , more simply, what a nanoscale is. Let’s deal at first with inorganic nanoparticles such as gold nanoparticles : t his material gains some specific physical properties only when the size of th ose particle s is extremely small – only few nanometres , like 5 nanometres , maybe 10. =f we overcome the 100 -nanometre threshold , it's going to be just gold. On the other hand, in medical field, anything that is smaller than one m icron is “nan o”. For example, if we consider microbes , their dimension relies on a range of 1 m icron : if we go below , it is possible to find anything that potentially could be called nano, so this definition may vary depending on which field we are considering. Nanomedicine i s a highly multidiscip linary field. So, we gather different backgrounds including physics, chemistry, biology, and, of course, medicine and engineering. Nanotechnology has been applied in a variety of different applications. First , we can do diagnostics : as nanoparticles can re ly on a very high mobility (due to their low dimensions), they can be potentially injected into our bodies through the bloodstream. And these particles can be used as a tracer for detecting specific diseases , depending on the physical properties of th ese parti cles . One main research field is about nano diagnosis, which concerns introducing inorganic nanoparticles in our body , and these nanoparticles are detectable , for example , through MR=. So , they need to be detected by so me sort of physical device. And then , if these particles can accumulate selectively on a specific tissue (such as a tumour tissue ) it is possible to find a very powerful way to loca lize such tumour site according to thi s cumulation of nanoparticles . :oweve r, one of the main areas of application is drug delivery : this takes inspiration from the “historical ” use of some liposomes containing chemotherapeutic agents to enhance the ir activity to target tumour cells in a more specific way , w hile minimising /decreasing the side effects of this “chemical robots ”. And , in the last 20 years, this field has been evolved in proposing different strategies in order to target selectively specific cells or tissue or organs that we want to treat , or to e xploi t the gene del ivery mechanism . One of the main areas of nanomedicine is developing nanocarriers so that car rier particles (still at nanoscale ) can encapsulate and track nucleic acids, which might be DNA or RNA , depending on t he application . =n principle , these particles can enter specific cells , delivering the genetic code in a similar way as the viruses do. And there , the new gene can be expressed /translate d into messenger RNA , producing/e xpressing the pr otein s we want to express. A very “f amou s” area of concern is represented by vaccines , but these are based on lipid nanoparticles, which encapsulat e messenger RNA : t hese p article s will be deliver ed and recognised by immune cell s, which can get these RNA in, and that will translate, induc ing the expression of spike pro teins (but it can be any sort of protein) , which then will be presented to the immune cell s so that the adaptive immune system will recognise this protein as a foreign body and therefore it will learn how to attack any sort of virus es or bacteria . Last example is on tissue engineering . Let’s think about the possibility of regenerating tissues : t here is a d amaged tissue somewhere , which can be th ought of as a cartilage defect somewhere in our joint . One way to treat it could be the injection of a hydrogel : s uch s ort of smart material, once it's made on the cycle def ect , will induce the regeneratio n of the tissue by inducing cell migration, proliferation, differentiation and so on until the cells themselves will start producing their own cellular matrix , basically reconstituti ng the healthy tissue. All around, indeed, if you want to achieve a sort o f smart material (smart hydrogens in this case ), the material has to be very well defined at the nanoscale in order to communicate with the surrounding cells through biological messages. Few examples of the use of nanotechnology in medicine: 1. Nanobiotechnology : This term refers to the application of nanotechnology to biological and medical fields. 2. Nanotherapeutics : This term refers to the use of nanoparticles and other nanoscale materials for therapeutic purposes. 3. Nano -encapsulation : This term refers to the process of enclosing a drug or other substance within a nanoparticle for targeted delivery. 4. Nanoparticle -based drug delivery : This term describes the use of nanoparticles to deliver drugs to specific cells or tissues. 5. Molecular na notechnology : This term refers to the design and construction of molecular - scale machines and devices for use in medicine. 6. Nano -scale drug delivery systems : This term refers to the use of nanoscale materials, such as liposomes or dendrimers, to deliver drugs to specific locations in the body. 7. Nanostructured materials : This term refers to materials with a nanoscale structure that can be used in medicine, such as nanofibers or nanotubes. :owever, sometimes the prefix “nano” is chang ed with something els e. This is just due to a research necessity, as several funding agencies started to get bored of such prefix , so nanomedicine has started to be called with some synonyms: - Molecular medicine emphasizes the use of molecular -scale technologies and techniques to diagnose, treat, and prevent diseases. - Precision medicine highlights the focus on personalized and targeted treatments that are tailored to an individual's unique biology and medical history. This term can encompass a range of approaches, including those that rely on nanoscale technologies, as well as other mole cular and cellular strategies. - Theranostics refers to the integration of diagnostic and therapeutic functions within a single system or agent. This approach involves the use of advanced imaging and sensing technologies to identify disease targets, as well as targeted therapies that can be precisely delivered to those targets to provide maximum efficacy with minimal side effects. According to some journalists, nanomedicine is something like developing n anorobots that are so clever that they can identify a specific cell on ce inject ed . Then, they can fix the cell /the tissue like a nano surgeon operation . :owever, if those nanorobots would actually exist, they would be attacked by the immune system as it would recognise them as foreign – and, conseq uently, unwanted – bodies. On the other hand , the real aim of nanomedicine is to r educe of dealing with materials bigger than one m icron . So, we can deal with pr otein aggregates or self -assembled polymers and pr ot eins, which give something big ger, but still below th e micron size range. =n this area, we can also find viruses which have particles in the range of 10 -100 nm . And from a physical point of view, these viruses are definitely nanoparticles, but they are extremely “cleverer ” system s from a “machinery” point of view, as they are able to b ind s pecific cells through a specific receptor and then , they will induce internalisation. So , virus es update in a way such that the nucleic acid will be delivered at some point , a nd then they are also progr ammed to allow this nucleic acid to enter the nucleus , and then, to induce its expression. This b asically consists in the coding and expression of new pro tein s that belong to the virus itself. So , a new virus will be formed and then it's also programme d in order to infect other surroundings . =f we can catch the biological or physical chemical language of this virus, one should be able to de sign the materials to let them do the same, but fixing cells rather than affecting them, despite the fact we are really far away from here. One example concerns nanoparticles m ade of lipids , which encapsulate messenger RNA, so they make sort of nanoparticles. And once these particles are internalised by new cells , the messenger RNA will use the expression of a certain pro tein , the Spike protein , which w ill be then present at the surface as an antigen. Such antigen will be recognised by the other cells such as the T-cells, which will later induce expression of antibody and the specialised T ce lls to attack this virus . Th ere is something we can already do, as we have some chemical tools to design and produce nanoparticles and nanocarriers which can be used for therapeutic application s. The oldest example is the liposome , which basically represent s the first nanomaterial used for drug deliver which has shown to be efficiently working . Since that moment, more and more nano medicinal drugs have been employed for clinical use , but the 90% among them a re still based on lipogenesis. Speaking of such particles, t he ir size is about 100 -150 nanomet res, and they have the characteristic of being made by a bilayer of phospholipid s with a liquid core . Such liquid phase has the same composition of the outside, which consists in the user buffer or physio logical fluid , a nd the drug can be entrapped within this liquid core. On the other hand, the external membrane is relatively unstable , so at some point , if th ose particle s accumulate at the site of interest, this drug would be released . Then , we can also do somethin g d ifferent : for example, we can use this “switch ” to have a therapeutic moiety , which can be a small molecular weight drug or a therapeutic p rote in, and such moieties can be protected by the presence of a polymer . This pol ym er will allow to preserve the activity of the drug during and after the injection into the bloodstream. =t's very important because as soon as you introduce any sort of a foreign material into our body, the immune system will generally recognise it as a foreign b ody and will attack and we'll get rid of it as soon as possible. :owever, it is possible to make a step further and synthesise and prepare full polymer particle s. The polymer can be of different type : for example, a nanoparticle has typically a hydrophobic polymer as a core, and this is useful because the hydrophobicity of the polymer will allow a self -assembling inside, other than easing the encapsulation of hydrophobic drugs (as most chemotherapeutic drugs are hydrophobic as well). :owever, even the outside layer has to be programmed in order to make it hydrophilic , i n order to provide the colloidal stability and to preventi ng the recognition by the immune system. Regarding the architecture of the pol ymer, there are some chemical tools, particularly in polymerization techniques, which are related to the controlled living polymerization , which allow to p rovide a very well -defined topology to the polymer , which can be useful if we want to encapsulate dr ugs as well as modifying the surface of the nanoparticle in order to target specific s ites. Then , we have to think about how we administer th ese nano parti cles /na nocar rier s into our body as there are different possible route s. The mo st commonly used is the intravenous injection , b ut we can use either other approach : for example , we can inject it intramuscular ly or typically we can use the intraperitoneal cavity to delay the release of some drugs. We can also inject a polymeric precurso r into a tissue defect and then to induce ge lation (which also represents an excellent example of tissue engineering) . So, there's a polymeric liquid precurs or which can be mo dified , so we can inject it exactly where we want to let them later interact with the cells. The big area of research comes from this basic idea of injecti ng some nanoparticles containing a chemotherapeutic drug, and if we are able to accumulate th ese particles exactly where the tumour is , we can enhance dramatically the effect of the chemotherapeutic agent. As we a re reaching a very high drug concentration locally, we can limit the side effects of this chemical at the same time : as we know, chemotherapy might be very efficient in killing tumours, but unf ortunately all these compounds are also toxic for healthy cells . So, if we exploit a vector/ carrier that selectively release s the drug exactly where we want , w e have a mechanism and effect, which is ca lled enhanced permeability and retention effect (EPR). =ts principle relies on the fact that , as the tumour tissue is highly disorganised and th at it needs to grow, regardless of how, when , and where , it need s a lot of blood to transport the nutrients. So , the EPR effect relies on the fact that the growing vascular tissue , t hat will go all around, is leaking , a nd this vasculature will be full of something like tiny holes, because of the defects in that forming layer on the endothelial cells of the vascular tube . Conseq uently , if we have some circulating nanoparticles in blood , they can passively accumulate in the tumour tissue because the y are escaping the blood flow entering this fenestra and accumulati ng in there (enhanced permeability ). But then , once they accumulate in there , t hey don't find a good way to go back to the blood stream (which, in a standard healthy tissue, may happen because of the presence of the lymphatic system ). Fabric vessel will drain the “physiological food ” that you find here , and they will give all this stuff back to blo od , but in this case , when you have a growing t umour , this lymphatic system doesn't properly work . Therefore, once the particles are in there, they will stay there for a while , a nd this is the part which is called reten tion effect . The full mechanism is a passi ve way in which nanoparticles can accumulate , so, if they can accumulate in this tissue, t hen they can release the drugs they have embedded at the be ginning. We have nowaday s a variety of different nanoparticles developed via polymers, lipids , inorganic material . =f we wonder h ow much nan omedicine for drug delivery will reach the clinical stage and , in some cases , h ave been approved by the regulatory authorities , s uch as the F DA , we find out that m ost of them are made of liposome s. The advantage of liposomes is that they are highly biocompatible materials, becaus e our cellular membrane s are made - up of phospholipids , so they are safe ; furthermore, they are relatively simple to be processed , and the regulatory authorities would relatively easily allow to make some clinical trials , whereas this process will definitely be much more complicated for fully synthetic m aterials . Another application which is widely used in phar maceutics concerns protein -polymers conjugate s. =f we have therapeutic proteins that potentially can treat a specific disease , t he problem is that , as soon as you inject these therapeutic proteins in our body, they will be easily recognised by the immune s ystem. =n our blood we have some enzymes, proteases , that will degrade t hese proteins quite fast . M oreover, if these proteins are too small from a hydrodynamic point of view, they can be excreted easily by kidneys. So , the only way to prevent all these unwanted effect s is to attach either a protein or a polymer which will make it bigger , enhancing the circulation in the bloodstream because th ey cannot be excreted by kidney anymore if they are big enough , :owever, at the same time, if the polymer is well engineered, you can also avoid the recognition or at least minimise the recognition of the immune system and the attack from the proteases in blood. So, a typical approach for producing new biotherapeutic agents is to attach polyethylene glycol (PEG) , which is a polymer that makes this job very effective. We can attach a PEG chain to a specific group of the amino acid sequence , and we get sort o f polymer surrounding the protein, which remains active , while the polymer preserve s its stability and activity. There are even drawbacks , which may be overcome by proposing alternative mechanism of polymerization in order to grow specific type s of polymers , wi th a specific chain length , molecular weight and topology of the polymer. There are many different therapeutic proteins that can be attached to the polymer that reach clinical trials and the market as well. Concerning inorganic nanoparticles , a wide field is represented by suprapara magnetic ones, which are made of iron oxide. They find nice application in the field of magnetic resonance imaging (MR=), as they can be easily detected and used as a contrast agent . :owever, there are several other inorganic material s which have been widely used. One of them is quantum dots . Quantum dots are kind of nano semiconductor material , a nd they have the intriguing characteristic that once they are illuminated with a certain light at a specific wavelength, they can emit new light at a different wavelength, like a fluorescent signal . So, th ey can be used as a very stable fluorophore in a variety of in vitro applicatio ns for characteris ing the specific activity of cells . By the way, these are toxic m aterial s, so they cannot be used for clinics. Then , another example concerns gold nanoparticle s, which have an intriguing character istic , which is based on the surface plasmon resonance . Because of that , when they are illumi nated with an incident ligh t, they can absorb this radiation at specific frequencies , allowing a change in the colour of the suspension, which usually depends simply on th e size of the particle, and this is a very interesting characteristic which has been widely used in medicine. At the same time , t hey are very good contrast agents in electron microscopy , so they can be seen very well as, for example , the black spots in the TEM image. Another very important characteristic is that they can release a lot of heat once they are illu minated by an incident light , and the generated heat can kill the surrounding cells , so this can be exploited for selectively killing tumour cells if we are able to accumulate nanoparticles in the tumour and then to find a way to illuminate this area with a certain light. The heat will rise the local temperature in a range of about 42 °C at which the tumour cells will die, despite this can even dama ge the tissue locally in order to get rid of the tumour area. All the design ed organic particles can be functionalized at the surface with some moieties like antibodies or peptide s, which allow sort of a communication with cells. Concerning tissue engineering application s, we can inject Polymeric resources in order to make a gel : This can be Done by exploiting specific properties of the polymer. For example, we can deal with polymers which are thermosensitive or therm orespons ive, Which means that , at low Temperature , like fridge temperature or even room temperature in some cases , These polymers are soluble in water or in a buffer . As soon as the temperature of the system rise s (for example, at physiological temperature of 37 °C), We have a temperatur e transition which is technically called lower physical solubility temperature . Because of that, the polymer p recurs or lose s its solubility in water , a nd it will start forming crosslinks, generating a network between all these polymer chains and the resulti ng three -dimensional network will be a gel or a hydr ogel , w hich means it's a polymer network surrounded by water. Furthermore, this polymer can also be functionalized with other biological moieties (polymers, peptides), in order to allow communication between cells and the tissue regeneration. We have mainly 2 possible approach es : You can either develop a sort of acellular hydroge l m atrix , which means that all the pr ecursor you're injecting i s just polymer and buffer , Bu t we may Also think in order to enhance this regeneration of the tissue to entrap some cells within the ma trix . So , we could also disperse a certain number of cells in this li quid pr ecursor, which can be engineered cells or even some specific cells that ca n induce a faster regeneration of the tissue , such as stem cells . The design of the polymer material at the nanoscal e is more complicated than that , because this should also allow adhesion to the cells inside of it , a nd then you also have to take the proliferation of the stem cells into account . Recalling the EPR effect, it is a very promising approach for accumulating nanoparticles in a tumour. :owever, it has been demonstrated that such accumulation effect is often not working wit h humans . This discrepancy with research paper is mainly because of the animal model used in research , in which the tumour is induced to grow way faster than a tumour which actually grows in human body . So, the main difference is that a tumour which is gro wing in an animal ha s a very highly leaky vasculature , and in there early the particles can accumulate easily , but when you shift that technique to humans , such vasculature is usually not so leaky as expected or as required. Th is represent s a huge problem in nanomedicine because anytime you want to develop a new nanomaterial you have to pass through animal models , which are very different from what we have in humans . So, in most cases, unfortunately, the clinical trials fail . The alternative would be to decorate Our nanoparticle with ligands that specifically bind receptors on the cell , which may be, for example, a cancer cell. Let’s think about antibodies : =f we definitely find a monoclonal antibody that specifically bind s the receptor on th e cell , if we link t he antibody to the nanoparticle, this w ill encounter T cell s at a certain point, and stick onto it as an antibody typically does. Once the particle spots the cell , it can release the drug which was encapsulated in close proximity : This results to be way more efficient than Passive accumulation such As the EPR effect. :owever, t here are big drawbacks in here as well, and they are related to the recognition of the immune system : as soon as you inject an y nan oparti cles =nto the bloodstream , There is a mechanism which is called op sonis ation . =t Basically regards The physical absorption of proteins at the surface of th ese nanoparticles . Opsonization is the process by which a foreign organism (a particle) becomes covered with opsonins . Opsonins are defined as any blood serum component that aids in the process of phagocytic recognition (complement proteins and immunoglobulins are the most common) . The opsonisation makes the foreign materials more visible to phagocytic cells, and phagocytosis can occur, which is the engulfing and eventual destruction or removal of foreign materials from the blood stream. This is the main clearance mechanism for the removal of undesirable components larger than the renal thresh old limit from the blood. :owever, =n the case of nanoparticles, clearance is typically carried out by the mononuclear phagocyte system . =t is represented by a set of different organs , Such as Liver , spleen and a few others, including also l ungs and kidneys. liver and spleen are full of immune cells, which are designed exactly for this jo b. for example , you can find the Kupffer cells in liver , And these are Macrophages that , as soon as they find opsonized particles, they will phag ocyte them. the consequence will Be that , as Soon as you inject nano particles, you get opsonization in blood and , after a circulation of few seconds , All of them will accumulate in these organs , and they will never reach the target. To overcome such problem, it is possible to decorate these nanoparticles with a polymeric layer which is known to be protein repellent , which means that it prevents the absorption of proteins at the surface. Basically, we decorate the surface of this nanomaterial with the polymer chains, which are highly hydrophilic , so they are able to structure a lot of water all around , and they will form a sort of physical layer to prevent the absorption o f the pr ot eins to the surface of the particle . as a result, you limit or prevent op son ization. The so -obtained particle is generally called stealth particle , w hich means that it's going to be invisible to the immune system. The gold standard material for making nanoparticles is polyethylene glycol (PEG) . This is a polymer which is highly hydrophilic , and dramatically enhance s its circulation in blood, so , if you have a nanop article th at would be op son ised by our body, it can circulate even for days, if not weeks , if you PEGy late it . =t's very important to define the grafting density of the polymer and the length of the polymer , which means , in other words, to identify its molecular structure. Furthermore, it has the advan tage of being functionalized at the termin i of the chain , s o we may also think to attach the targeting mo ieti es (like an antibody or a specific peptide or ligand ) after the PEG ylation , in order to achieve that active targeting. W e have many options : we can use an antibo dy , which can be link ed somewhere via some chemical reaction , but, on the other hand, we also have to think about the size of the molecule , which is relatively big: its hydro dynamic diameter is about 10 nanometres or , sometimes , can be even as big as the nanoparticle itself. =t is possible to use smaller molecule by linking only part of the antibodies : this is done by using an enzyme called papine , which selectively clean part s of the antibody to bind just its tiny fraction to th e nanoparticles . Furthermore, we can identify specific peptides, which are relatively short sequence s of amino acid s (if compared to a protein ), and that can be specific for binding a receptor as well. So, we have the advantage that we can synthesise an antibody ourselves using peptide synthesis , and then we conjugat e this antibody in a relatively easy manner . there's also another trend nowadays , which is related to the use of aptamer s. A ptamers are nucleic acid (DNA or RNA ) sequences tha t can self -assemb le and bind specific receptors and cells similarly as an antibody does , w ith the advantage that they can be shorter, they can be synthesised , and they can be also easily conjugated with the nanoparticles. We can even use micelles , which are simply a surfactant , with a hydrophobic tail and then a hydrophilic head. =n the case of nanomedicine, the material has to be biocompatible , so phospholipid s could be a good exam ple . Lipids, by the way, are widely used in nanomedicine, not just for making micelles , but mostly for making liposomes . They are very interesting and intrig uing material because they can form a water -based core , where therapeutic molecules, even proteins, can be encapsulated. Solid lipid nanoparticle s a re synthetised by melti ng the lipid p hase, creati ng a m icro emulsion : Once you get the nano droplets, you cool it down and these nanodroplets will become nanoparticles , and they are widely used for encapsulating hydrophobic drug s. Concerning polymers, it is possible to use a block copolymer : the simplest linear one is generally defined as AB block copolymer . One block is hydrophobic, while the other one is hydrophilic , other than biocompatible, and this polymer can self -assemble , forming polymeric micelles , which are us ed for encapsulating drugs in a similar way as the more commonly used low molecular weight surfactants. Furthermore, we can also prepare polymeric vesicles by means of a tri -block copolymer : if we call this B AB or ABA , we have the block in the middle which is hydrophobic , while the other two terminal are the same , hydrophilic block . =n this case, such block copolymers can self -assemble , forming a vesicle in a similar way as liposomes are formed by phospholipids. This, of course , presents similarities with liposome s, but the polymeric vesicles result to be much more stable , because there is a much stronger hydrophobic interaction between each hydrophobic block, so we have more interactions and the structure is packed in a way to be more sta ble , but stability can be an advantage or a disadvantage depending on the application . =t May be an advantage because , for example , the more stable they are, the more they can circulate in blood , but , on the other hand, if you have to release a drug, which is encapsulate d here, it takes longer to be released. Furthermore , the disadvantage of such a structure is its synthesis, as we need to synthesise a triblock copolymer , which is fully synthetic and, generally, the synthesis itself is not straightforward : it is Kind of challenging to get exactly all the same chains having tr i-blocks and having at the same time exactly the same molecular weight. Concerning homopolymers instead , We can do a lot of othe r things : Let’s consider a simple polymer with A chain , Composed of repeated units Of the same monomers. The most interesting application of such simple polymeric structures concerns the formation of the so - called polyplexes : several times, we need to enca psulate a nucleic acid , such as DNA or RNA , which is more or less a polymer , and it is negatively charged. Because of this, such Macromolecule s can be encapsulated with a positively charged polymer – a polycation. =f we simply mix the two things together i n water, we have a complexation because of the electrostatic interaction , so you generate a nanomaterial with the nucleic acid, which is complexed with the positive charges of the polycation here. depending on which axis you use , we :ave a positively charged polymeric nanoparticle at the end , with some DNA or RNA to interrupt this coil : this nanoparticle is called polyplex , a nd it's used as a non -viral vector, which means that we deliver nucleic acid in cells without using viral vect ors. The other technology would be to take a virus , remove the original DNA or RNA, and reintroduce the nucleic acid we want to deliver, and then we use the capsid of the virus to penetrate the cell and to deliver th is nucleic acid. =n this case, instead, you use a “known viral vector ” which is a polymer synthesised by you. Lipoplexes consist in the same thing basically, but instead of using a positively charged polymer, a polycation, = could use a positively charged surfactant, having an hydrophobic tail And a hydrophilic head which is positively charged , and nanoparticles are formed still by complexation with DNA or RNA. There are some advantages here. For example, the RNA vaccines used now for COV=D Are kind of lipid particles, so they are based on lipop lex es instead because , From a biocompatibility issue, it's much easier to deal with the phospho lipids even if they are positively charged, so they are slightly more toxic than the negatively charged one, but they can be still easily Metabolised or released by our Organism . Another category of materials concerns inorganic nanoparticles . The inorganic nanoparticles are mainly used Fo r diagnostic, even though They have been used for drug delivery as well. The limit of inorganic materials for delivering drugs is the fact that , if we have a solid material Which the nanoparticle is made of , the only thing that you can do to encapsulate th e drug is to Adsorb it all around the surface , But this Surface has a limited number of molecules that can be adsorb ed, even more if compared with the inner volume, which allows to have much more space. The other limitation is intrinsically related to the first one: if we have a biocompatible particle , it doesn't trigger any op sonis ation. :owever, if you admit Drug molecule to be adsorbed onto such surface , the final particle is no longer as stealth as before , because now we have a different surface property , and opsonins can now recognise this new particle as a foreign body. So, it is much better to us e the properties of an inorganic material for diagnostic instead , because we don't need a drug to be encapsulated. There are different possibilities concerning carbon -based materials , which are very intriguing (nanotubes , graphene) , because they have a lot of interesting physical properties but, on the other hand, they are very toxic , because our body is not able to degrade them very quickly. Gold nanoparticles are definitely safer , because g old is a typical example of inorganic biocompatible which doesn't trigger any immune reaction. On the other ha nd, it is not bio degradable. So, if you think of injecting nanoparticles in our body , these nanoparticles will stay in our tissue for really long times, potentially trigger ing some sort of long term nanotoxicity side effects. Calcium phosphate nanocomposites are better , simply because th ese are now made of inorganic materials, which are really biocompatible , and calcium phosphate is something that you can find in our bone s, so this kind of material can be used in principle for biomedical applications. Furthermore, even silica nanoparticle s have already shown to be biocompatible : silica is pretty famous because it form s a kind of network between the silicon atoms and oxygen , which allows O: groups to be shown all around the surface of such sort of nanoparticle s. Such groups are highly hydro philic, and they don't generally trigger the absorption of opsonins , Or at least, even if it does, the final consequenc e doesn’t involve the activation of the immune system . =n principle, if you think about the solid silica, it is non degradable, but if you deal with the silica gel (which means a gel made of kind of 90% water and just 10% or even less of this material ), it has been demonstrated that , under physiological cond itions , this structure can be slowly degraded by hydrolysis , so silica may find applications from this point of view . So, it is possible to say that nanomaterials allow a wide range of possibilities, as it is possible to vary the composition of the core ma terial , as well as the composition and , therefore , the final surface properties of the nano material itself and its surface. =ndeed, it is important because the interface between the nanomaterial and the surrounding biological environment regulates several aspects such as the kinetics of release of a certain drug . Generally speaking, there is an optimal concentration range when you deal with the drugs , and We can generally identify a lower and an upper concentration limit : clearly, when the concentration of the drug is too low, we don't have any therapeutic effect. So, if we inject a very low amount of drug in the system , the cells don't even respond or respond not really sufficiently. Then , there is a range where we really have therapeutic effect , and the concentration is kind of optimal to get a nice response by cells. :owever, if the concentration is too high, this drug start s being toxic. =deally , when you inject a drug in a body of a patient , You would like to get A constant concentration over time , which stays at the optimal range of concentration. Then, sooner or later, this drug will be metabolised or released by ki dneys , but you get a very long or prolonged effect anyway. however, the things Do not go like that really often : if we think about simple injection , there is a very rapid increase of concentration in the blood , so you may overcome this upper toxic limit , but then you dilute immediately this drug in all the blood volume, so you get a low concentration of the drug very soon . This is not optimal at all , as there is a range of good concentration in a very short time. So , when we deal with pharmaceutical formulations, we would like to achieve a sort of sustained release of drug , and this can be achieved in many different way s, such as multiple injections , But this is Oft en non ideal as there are nanoparticles that keep on circulating in our blood for a while because they are stealth , And while they are circulating, they slowly release our drug. The other important aspect is typical of tissue engineering : when you implant a certain gel at the site of interest, we want to release a therapeutic agent as well , And there's not only a question of sustained release, but you also have to consider The volume where this gel is located. So , there is not only to consider the concentr ation in blood, but also The concentration in a certain space , and the problem can be Slightly more complicated even from a mathematical point of view . Focusing on phospholipid s, they have quite an intriguing structure , even because their hydrophobic tail usually consists of two different tails , and One of them often contains a double bond which is planar : this means that it allows to freeze the structure of this tail , And this is very convenient because , when they self -assemble , there is the form ation of the typical Bilayer of lipids , either in liposomes and also in cellular membrane , for example . The great characteristics of this is the possibility to form a kind of zip because of this specific architecture of the lipidic chains , so the final structure will be much more stable. When we deal with liposomes , we form these spherical bilayers from lipids which can encapsulate nucleic acid in the core or even nano crystal s or, if we have a hydrophobic drug, you may think of encapsulate it by trapping it in the hydrophobic part of the of the bilayer. Then , concerning biomedical application s, the liposome is generally pe gy lated or , at least , there is a coating of hydrophilic polymer which enhances the circulation in blood because it avoids op son isation. Other polymers which are employed in nanomedicine are - Polyester , which is already widely used in clinics . =t's a biocompatible polymer with the great advantage to be biodegradable under physiological conditions , s o its ester bond can be hydrolysed . So , if we have a suture and we use this kind of material, the polymer will be dissolved simply by waiting. The other advantage related with nanomateri als is that we can prepare polymeric materials, including this polyester , with the great advantage that it does not accumulate in our body , so there is no risk for our health in terms of the materials we are using . furthermore , we can make an AB block copo lymer, where the B block, the hydrophilic one , is PEG , while the A block can be a polyester which is hydrophobic instead. So, it is possible to create micelle s or , in other words, polymeric nanoparticles for therapeutic injection . We have different commonly used polyester because a var iation in the type of repeated units allow to vary the degradation rate of this material : the longer the hydrophobic chain is, the slower the degradation will be. - Cellulose , which Can be extracted from plants. - Chitosan is widely used as it comes from the ch itin , which is a natural polymer which is extracted from shel ls of shrimps or crabs . according to a hydrolysis process , this chitin will be converted in c hitosan , and it presents lots of N: 2 groups : these are amines , positively charged group s, so the c hitosan itself is a polycation , and it is the typical example of positively charged polymer which can self -assemble , for example with nucleic acids as they are negatively charged . Moreover , it's biocomp atible, so it doesn't trigger too strong immune reaction s. - Starch has been used for biomedical applications. - Hyaluronic acid is a polysaccharide, as the other mentioned polymers , b ut it is one of the major components of the extracellular matrix in our body , which is mainly composed of collagen and hyalu ronic acid. So, the injection of hyaluronic acid won’t trigger any immune rejection , So =t's absolutely biocompatible, but there is a strong disadvantage, but it would be an advantage as well : it's very easily degraded by enzymes which are present in our body. :owever, we are also full of hyaluronic acid both in blood and in our tissues, and therefore , after a while , the hyaluronic acid will be degraded. So, this can be a big limitation in nanomedicine , because it may be that you prepare this nanomaterial, you inject it in blood and , after a few hours, the enzymes degrade it, so you get rid of it, and we don't get the nice release of a drug where you want . - Alginate is another natural material which is extracted by seaweeds. =t's a polysaccharide and a polyanion, and it's full of carboxylic acid groups . =t is fully biocompatible , but not biodegradable as hyaluronic acid. So, we have the opposite problem here : =t doesn't degrade but it stays in the body for really a long time , and there could be long safety concern s. :owever, it is interesting as it can easily make gels by complexation with calcium. So, the calcium is an ion whic h is divalent , while the polymer is negatively charged , so they will itself assemble in a very order ed structure forming the so -called calcium alginate, which is considered to be a. Kind of standard material, for cell microencapsulation. - Gelatin is a prote in-based material which is widely used as well, because it's biodegradable and natural derived. Several polymers can show specific, intriguing physical chemical properties depending on the way they are synthesised. Polya crylamide is an example of thermosensitive polymer , which is fully soluble in water, but at relatively low temperature. So , it can be dissolved pretty well at fridge temperature , but at 37 degrees the polymer is not so soluble as before because there is a thermal tran sition (lower critical solubility temperature ) because of which the polymer will tend to precipitate , form ing a gel. Concerning tissue engineering application s, this is something that we are looking for very often: we can take a cold liquid, inject it at t he site of interest , and because we reach the physiological temperature, there is a fast gelation, and we form instantaneously a gel where ver you want. So , this is a kind of phase transition which is triggered by an external stimulus that in this case is the temperature. :owever, we can play with different stimul i: There are polymers that are sensitive to ultrasounds, magnetic fields, oxidation , which is a quite interesting application as well. Thi nk about cells that are under inflammation : because of this, these cells release the so -called reactive oxidative species . These are substances that have strong oxidation potential , so, if we design a polymer which respond s to oxidation , it will be degrade d because of the presence of these reactive oxidative species , allowing the release the drug exactly where we want , as soon as the particle reach es that tissue because of the inflammation . Furthermore, t here are polymer s responsive to light electricity, me chanical stress, p: . This is another intriguing application : m any polymers can be designed to be p: responsive to make them to be not soluble in water when the p: is relatively high , under basic condition , but soluble when the p: is lower. This can be expl oited as there are tumours where the local p: is slightly acidic (6.8, which is lower than the physiological p: of 7.4) : as soon as the particle s reach this place , the p: drop s down a little bit , allowing the polymer to be dissolved , and the drug will be released only there. Enzymes are interesting if we have polymers with some sequences that can be cleaved by the activity of certain enzymes : we can use this stimulus to trigger the release of certain drugs only where the enzymes are p resent. in some cases, tumour cells overexpress metalloproteases : These are enzymes which are released generally by cells to degrade the extracellular matrix. Tumour cells want to do so because they want to undergo metastasis , so they need to make space all around the extracellular matrix, so they release a lot of metalloproteases (the so -called MMTP ), and this enzyme can be used if we design a polymeric material which can be degraded by this enzymatic activity. A possible way to prepare polymer nanoparticl es concerns the dissolution of such polymer in an organic solvent, which could be dichloromethane , acetone or any organic solvent which can dissolve the polyester. Then , we can mix it together with a hydrophobic drug and we can make a nice solution containing both components , and then we add the water phase with a surfactant , which is important because you need to emulsify this organic phase. Then, droplets are formed upon sonication (or even just s haking in some cases): the organic phase represent s the core, and it contains the organic solvent, the hydrophobic polymer, and the hydrophobic drug. The surface of this nanodroplets is stabilised by the presence of the surfactant , which has a hydrophobic tail and a hydrophilic head : this latter one stays on the outside, stabilising the spherical surface of the particle. Then , we can remove the organic solvent simply by evaporation. We can use a rotavap or, which is a machine used for evaporating this organi c solvent under vacuum. At the end, only a suspension of polymeric nanoparticles in water will remain . :owever, there is to consider that the surfactant that you need to use in this process may often not be so compatible as expected. So , we can also think of using the block copolymer as a surfactant or a stabiliser . For example, if = use a polycaprolactone , this block copolymer can be used for stabilising the surface of my polymeric nanoparticle , because , at the end , = have the polyester with this copolymer which are assemble d in this way with this hydrophilic block sticking onto the hydrophobic polyester and the peg block, which is hydrophilic, likes to stay at the water interface. at the end , it is also very conveni ent because you basically have a p egy lated particle straight away , and PEG is generally needed to avoid o pson isation, so you already created a sort of stealth polymeric nanoparticle simply because you use this block copolymer instead of a simple surfactant . As we already said, we can generate polymeric micelles by using block copolymers , but We can also think of functionalizing the end group of this polymer with a ligand such as an antibody or a peptide for cell targeting. We already introduced the import ance of identifying the critical micellar concentration , which represent s the major drawback in Nanomedicine regarding the application of micelles for drug delivery : =t's typical of any surfactant block copolymer = can easily prepare a suspension of micelles with a drug encapsulated with , which can be potentially injected into the patient. :owever, once it is injected , you have to take into account that the volume of t he blood volume in a patient is about 5 litres . So if the micelles were formed, so definitely you were above far above the CMC , as soon As you inject this stuff into the body, you dilute it a lot and you dropped down at the concentration far below the CMC , And the consequence would be that you immediately dissolved this micelle suspension , So you release the drug straight away soon after the injection, and your drug will never reach the target. So, it doesn't work unless you find the way to decrease the CMC as much as you can so that , even when injected, the dilution is not so relevant. the best strategy to be used may be to design the so -called unimolecular micelles , which are made of single, already branched polymer, rather than a block copolymer . polymer conjugates are polymers Characterised by having sort of hydrophilic main chain (We call it main backbone ), But there are side chains as well, which are attached to the main chain . W e may have, as si de groups , Drugs , so we can have a drug molecule to be chemically linked through a double bond to this main backbone. We can also have hydrophobic domains. Other groups can be used a s ligands , such as peptide s for active targeting. At the end, you design A polymer with the main backbone , then you introduce a drug to be attached. Then , if = add a hydro phobic tail , as well as a hydrophilic one, what happens is that you have a self -assembling in water , and you may form nanoparticles with the hydrophobic domain in a core , And the hydrophilic parts staying as a shell , Presenting, for example, the peptide fo r targeting. =f the drug is hydrophobic, it will stay inside , and we can even think to remove the hydrophobic component and getting just a hydrophilic polymer which travels bloodstream and displays the drug , and this polymer can reach the target because of the presence of the ligand . Focusing on dendrimers , these polymer s are intriguing just because of the structure itself , but its synthesis is very complicated and costly : we start with a difunctional amine , you let them react with this acrylate to get a Tetra functional molecule starting from a difunctional one . Then , this group now can further react with the same molecule as the beginni ng . fin ally, you get still a Tetra functional molecule, but now you have an amine at each terminus , so you can keep on running this reaction many different times , and at each cycle of reactions you generate new branches. So, you can grow the size of the dendrimer as much as you want. the advantage of dendrimer is to have a very regular structure, so you definitely have a full control over the size of the nanomaterial of the polymer itself, and you also have a full control of the number of functional group s on the surface at the end. Such surface can be further functionalized with drug s, peptides and so on , b ut you have to take into account that each of these reactions m ust be very precise, which means that the conversion has to be maximised, because otherwise you get the risk that some branches can keep on growing and other branches they stopped because the conversion is not 100%. And the other pain is that , after each of t his reaction, you need to purify your material, getting rid of the unreacted species , a nd then repeating the reaction afterwards. So, considering the number of steps that you have to carry out at the end, preparing dendrimers is very costly. =t works prett y well on a lab scale, but considering getting dendrimers for Scaling up in industry, even in pharmaceutical industry, is not a great idea. generally, we rather prefer to deal with the hyper branched polymers which are something similar, but they do not ha ve the very regular structure as in the dendr im ers . Rat her than getting this nice spherical order structure, you get a Number of branches , but they can Still make some Kind of spherical coil s in water at least, which have a high number of functional groups . the big advantage is that the synthesis of them is much, much easier than the synthesis of dendrimers. One of the basic concepts of nanomedicine =s biodistribution : any time you design a new nano carrier, that is used to deliver a drug or used for detection and diagnostic , we have to figure out the fate of the particle itself . We already mentioned the importance of the mononuclear phagocyte system : when you inject particles in the body , they can circulate for a long time if they are pe gy lated , But t he y will be accumulated in organs and tissue s, and hopefully they will be accumulated at the site of interest as well. So, we have to study where this particle will be distributed in our body after the administration. Sooner or later , they will go to spleen, but it is even possible to see some material acc umulated at the tumour si te, which allows the sustained release of the drug where we want. :owever, there is to remember that kidney is a high vascularized tissue, and it is also specialised for getting rid of the nanomaterials. an importan t Area in nanomedicine regards quality assurance and regulation, which concerns dealing with the regulatory authorities : whenever you develop a certain new nanomedicine in the lab, our goal would be to reach the clinical stage And , after all, to get a commercial product. Clearly , this is very challenging , as it takes years of research : any sort of standard pharmaceutical drugs generally takes 10 years. Clinical trials involve people, so there are a lot of ethical issues regarding steps. We have three di fferent clinical trials : step 1, Step 2 and Step 3 (or phase 1 , 2, and 3) . more importantly, all these steps ha ve to be carried out according to quality assurance regulation , which means that we have to manufacture products according to certain quality and control strategies. in any case , when you deal with the manufacturing , if the final product doesn't work well, you can always throw it away and buy a new one. With nanomedicine product s you cannot think in this way , because you are dealing with the health of patients , as there's a lot of medical issues which can kill people because of a lack of quality control. =n other words , each step of this drug development process, including the manufacturing process, ha ve to fulfil the requirements of the quality iss ue, so this is a very important part Of nanomedicine , as well as any other pharmaceutical approach. 2. Diagnostics nanom aterials which might be used in diagnostics are designed to be traceable : once injected , they travel A little bit Through the bloodstream, accumulating in a certain area and in there we can visualise the particles and figure out what's going on in th at tissue . typical examples of contrast agent s in MR= , PET, C T scan concern the use of particle s in such a way that they are fluorescent , so they can emit light, and we can easily visualise them by using an optical detector. Unfortunately, even though this technique is widely used in vitro with cell culture s (employed to assess the biological mechanism of death ), it is qui te challeng ing to be applied: if we want to visualise a particle inside a body , we have to achieve light penetration into the patient. =f we have a particle which emit s light , it Can be detec ted, but if the light Doesn't pass through the tissue, it could not be visualised . =f we consider the wavelength of the light radiation , it is usually small (very close to UV region), corresponding to the region where the energy is the highest , Because the wavelength is inversely proportional to the frequency of the radiation , and this latter one is in turn associated with the energy of the radiation (And , of course, if you go the other way , at the threshold between the infrared light and the visible light, the energy in the area is much lower ). We may think to cho ose the UV light to allow a deeper penetration of light bec ause it involve s more energy. :owever, if the wavelength is close to th e UV , you can still have very small penetration, even less than one millimetre , while if we use near infrared light , the penetration depth can be much higher than four millimetre s. =n general, the maximum Depth within a tissue that = can reach is about 5 m illimetres, as the tissue clearly needs to stay alive , so =t's a very small thickness indeed. So , if we think about the nanoparticle which has been accumulating in the tumour area inside , we can n ever see them Because the light is not able to penetrate mor e than 4 millimetres. So, if you want to deal with fluorescent nanoparticles, this technique works well with small animals (such as mice for in vivo tests), But there's nothing to do with the clinical applications, unless the tumour or the disease you are dealing with =s close to the skin , or at least reachable with a sort of a device or detector. But why does the red light penetrate more than the blue light ? That's mostly because the melanin and haemoglobin , which is present in our blood, absorb a lot of light, particularly in the blue region , but this absorption slightly decrease s when the light is closer to the Red Sh ift , so it’s Better to use Near infrared light rather than blue Violet colour. So, the only thing that we can do with these fluorescent nanoparticles is to use them with the fluorescent microscopy in vitro cell s. =ndeed, they are widely used for this scope. Briefly, the working principle of a fluorescence microscope concerns the use of a specimen which contains your cells stain ed with some fluorophores, which are molecules that are able to emit light once they absorb light at a different wavelength. So, your specimen is illuminated with a light (typically from a sand lamp or a mercury lamp) =n a specific range of wavelengths , but not before such light has passed through one or more filters which allow the passage o f only a certain band of wavelength. Then , this li ght will be reflected with the mirror, and it will Encounter our specimen in there. The fluorophore will absorb the light at this wavelength and , consequently , the specimen emit s a light at a different wavelength such that we can distinguish between the light you're providing, and the light emitted by your sample. The light emitted by your sample will pass through th e so -called beam splitting mirror , and it will reach the lens corresponding to the Camera which can record or get the final picture of your sample. To understand how fluorescence works, let’s recall the fact there are certain molecule s which ha ve a certain ground state of energy of electrons in their structure. So, when they are illuminate d with a light at a certai n frequency, these electrons can jump at high energy levels, and then they will decay , emitti ng photons in order to go back to the ground state, so we release energy in this way. =f they do so, the photons they have emitted will provide the fluorescence . Generally, a typical fluorophore contains a lot of double bonds aromatic rings that will absorb this lig ht and according to a certain spectrum , so there will be a maximum at which at a certain wavelength it will absorb the most , and this energy will be release d as a fluorophore at another wavelength. the maximum of this emission factor is called the emission wavelength . generally, there is a shift between the peak of the absorption and the peak of the emission : in other words, this distance will tell you how far you are between the light that you use for exciting it and the light which is emitted. Clearly , this is also important