The past few years has witnessed a booming market of protein and peptide drugs, owing to their superior efficiency and biocompatibility. Parenteral route is the most commonly employed method for protein and peptide drugs administration. However, short plasma half-life protein and peptide drugs requires repetitive injections and results in poor patient compliance. Oral delivery is a promising alternative but hindered by harsh gastrointestinal environment and defensive intestinal epithelial barriers. Therefore, designing suitable oral delivery systems for peptide and protein drugs has been a persistent challenge. This review summarizes the main challenges for oral protein and peptide drugs delivery and highlights the advanced formulation strategies to improve their oral bioavailability. More importantly, major intestinal cell types and available targeting receptors are introduced along with the potential strategies to target these cell types. We also described the multifunctional biomaterials which can be used to prepare oral carrier systems as well as to modulate the mucosal immune response. Understanding the emerging delivery strategies and challenges for protein and peptide drugs will surely inspire the production of promising oral delivery systems that serves therapeutic needs in clinical settings. Figure 1). Recent developments in the biotechnology and pharmaceutical sciences have made it possible to produce potential therapeutic PPDs in commercial quantities Enormous efforts have been made over the past few decades to realize the therapeutic efficacy of protein and peptide drugs (PPDs). Owing to their excellent specificity and biocompatibility, PPDs can achieve ideal therapeutic effects at relatively low doses Though parenteral administration is the most commonly employed administration route for PPDs, it often associates with poor patient compliance In this review, we summarize major barriers for oral delivery of PPDs, and the state-of-the-art formulation approaches for promoting the oral bioavailability of PPDs. Intestinal cell targeting strategies are presented with an emphasis on examples that showed great potential for clinical applications. Additionally, multifunctional biomaterials which can be used to prepare oral carrier systems as well as to modulate the mucosal immune response are also discussed.The absorption of orally administered PPDs from the GIT into the systemic circulation is limited by various factors. These include the release of drugs from the carrier systems and pass on their way to the target receptors within the harsh intestinal environment. Ingested PPDs first encounter digestive enzymes in our oral cavity, including amylase and lipase in the saliva Figure 2 shows the mucus layer covering GIT epithelial membrane is considered as the first physical barrier. Mucin is the main component which is a highly glucosylated glycoprotein. The backbone consists repeating sequences of serine, proline and threonine residues. The O-linked oligosaccharide side chains are generally terminated in L- fructose, sulfonic acid or sialic acid. Therefore, the intestinal mucus layer shows negatively charged The two major mechanism of drugs permeate through the intestinal mucosa are the passive diffusion via the transcellular or paracellular pathway Figure , and theThe oral bioavailability of PPDs is often hampered by their physicochemical characteristics, such as hydrophilicity, large molecular weight and sensitivity to enzymes and pH. To alter the physiochemical properties of PPDs, chemical modifications strategies, including lipidization, cationization, PEGylation and prodrug formation have been applied.Rapidly and completely transported drugs are generally lipophilic and distribute readily into the epithelial cell membranes of GIT 10 and bile salts, presumably to promote peptide drug permeation. C10 elevates intestinal membrane fluidity via interaction with protein and lipids on the membrane, and it permeate over through both transcellular and paracellular pathways. However, Sakai et al. reported that high concentrations of C10 (>50 mM) could lead to significant cytotoxicity to Caco-2 cells, thus limiting the use of this technique One example is the leu-enkephalin peptide which is chemically modified by a reversible aqueous lipidization method with a dimethylmaleic anhydride analog. This resultant drug was stable in various pH phosphate buffers and showed greater stability against enzymatic degradation. The study demonstrated the lipidization may be an enabling strategy which can be used to enhance oral absorption Cationic drugs are more permeable over the intestinal mucosa compared with anionic drugs, it is due to the negatively charged glycoproteins and glycosphingolipids on the intestinal cell membrane Futamiet al. demonstrated the negatively charged mammalian cell membrane consisting glycoproteins and glycosphingolipids, cationization of these proteins elevated their ability for intestinal drug permeation Studies have showed that PPDs can be cationized by chemical conjugation demonstrated efficient intracellular delivery via adsorptive-mediated endocytosis. Generally, PEGylation is the covalent attachment of polyethylene glycol (PEG) to PPDs and elevate their half-lives due to steric hindrance against proteolytic enzymes. The increase in the molecular mass can improve both pharmacokinetic and pharmacodynamic properties of PPDs Minimolet al. have developed a PEGylated starch acetate nanoparticulate system for oral insulin delivery. An amphiphilic polymeric derivative was obtained by PEG conjugating with starch acetate, subsequently incubated with drug solution at the critical micelle concentration, forming self-aggregated drug loaded PEGylated nanoparticles. These self-aggregated nanoparticles showed only 32 nm in size allowing large surface area of the particles to contact with the intestinal mucosa. Moreover, the nanoparticles with great intestinal mucosal bioadhesion further promoted the drug permeation over the intestine  in vivo usually undergoes transformation either by a chemical or an enzymatic reaction. Esterification of hydroxyl, amino acid, or carboxylic acid containing drugs can increase lipophilicity, thus improve intestinal drug permeation A prodrug is a chemical derivative of a main drug, it usually has greater stability, solubility, lipophilicity and intestinal permeability. It converts to an active drugVerdineet al.Clarket al.in vivo antitumor activity Cyclic cell-penetrating peptides (CPPs) show high stability and demonstrate great potential for the intracellular delivery Absorption enhancers are usually one of a varied class of chemical moieties, they are used to improve drug absorption by facilitating intestinal cells permeation Sadekaret al. have developed an oral form of camptothecin by co-delivering with cationic, amine-terminated dendrimer, which is a promising intestinal mucosal penetration enhancer, drug solubilizers for oral drug delivery. The results showed camptothecin solubilization in gastric fluid and significantly enhanced oral drug absorption without opening the tight junction The commonly used absorption enhancers are surfactants, fatty acids, chelators, glycerides, bile salts, salicylates, chitosan and cholesterol. They normally increase the solubility and bioadhesion of the drug or drug carrier system which allows more drug amount to be retained at the absorption site and resulting in greater drug oral bioavailability Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a promising absorption enhancer can enhance passive permeation of polar charged drug molecules through the intestinal epithelium. This is noteworthy in view of the very low tendency of a polar drug to permeate over the lipophilic intestinal epithelial membrane N-2-hydroxy-5-chloro-benzoyl)-amino-caprylic acid) (5-CNAC) is the leading examples of Eligen® technology from Emisphere. It was reported that 5-CNAC can deliver macromolecules (> 150 kDa), enhances transcellular absorption without disrupting intestinal integrity. Karsdalet al. incorporated 5-CNAC with calcitonin for oral administration. 5-CNAC interacts with calcitonin forming an insoluble entity at low pH in stomach, once it reaches small intestine at higher pH, the complex dissolves and facilitates intestinal drug uptake, resulting in systemic exposure of intact peptide Another effective permeation enhancer, 8-, leupeptin , chicken ovomucoid (trypsin inhibitor) and FK448 (chymotrypsin inhibitor). These proteolytic enzyme inhibitors are usually co-formulated with PPDs to prevent enzymatic degradation in intestinal mucosa. However, it was also reported that the safety of using enzyme inhibitors is a major concern. The excess use of this excipients may restrict certain therapeutic effects or trigger undesirable pharmacological activities The most clinically advanced enzyme inhibition example is an oral insulin formulation known as ORMD-0801 consisting soybean trypsin inhibitor and a chelating agent that scavenges calcium. This treatment showed a significant 24.4% reduction in the frequencies of glucose readings >200 mg/dL, and a significant mean 16.6% decrease in glucose AUC Liuet al. have developed a novel self-assembled nanoparticle composed of insulin and trimethyl chitosan, and a dissociable mucolytic agent. The mucolytic agent modified nanoparticles exhibited free Brownian motion and facilitate drug permeation over intestinal mucosa. In diabetic rats, the mucolytic agent modified nanoparticles generated a prominent hypoglycemic response and showed an bioavailability of 2.8-fold higher than that of unmodified nanoparticles. Mucolytic agents, also called mucus penetrating agents, which are able to facilitate the permeation of the drugs across the mucus barrier and elevate oral bioavailability of PPDs Cell-penetrating peptides (CPPs) are usually derived from viruses that are efficient at cell entry or membrane translocation, non-viral proteins or smaller molecules normally interact with membrane glycosaminoglycans, promoting PPDs to enter intestinal epithelial cells via endocytic pathways. However, the use of CPPs to elevate the oral bioavailability of PDDs has not yet been validated in the clinic Kameiet al. have used oligoarginine as a CPP to elevate the oral bioavailability of the peptide drug, leuprolide, the results found that leuprolide-oligoarginine conjugate attached to cell-surface proteoglycans and subsequently permeate over the ileal epithelial membrane via endocytosis pathway Recently, CPPs such as HIV-1 Tat, penetration and oligoarginine are commonly used for oral delivery of various drugs Onishiet al. have developed enteric-coated chitosan-4-thio-butylamidine conjugate microparticles for oral delivery of calcitonin Yuet al. have developed a glucose-responsive microsphere that could be used as an efficient insulin carrier for oral delivery, and resulted in sustained hypoglycemic effect Microparticles (size varying 1-100 µm) with high surface to volume ratio and greater intimate contact of the drugs with the intestinal epithelial layer, prolong gastric resident time, thus lead to higher drug absorption and oral bioavailability Hydrogels generally contain water phase, a crosslinked polymer and a drug component. Usually they can respond to environmental changes to alter network structure, mechanical strength and swelling manner O'Neillet al. have developed whey protein hydrogels encapsulating riboflavin. The dried microbeads hydrogel showed great resistance to GI degradation, underwent swelling and sustained release drug in GIT Nanoparticulate carrier systems, usually with particle size of less than 1 µm, such as polymeric or lipid nanoparticles, nanoemulsions and niosomes for oral drug delivery are of interest owing to the great benefit in promoting drug stability, provide a sustained drug release profile and elevate drug absorption over intestinal wall. In general, smaller particles of less than 500 nm are usually undergoes endocytosis and shows greater intestinal drug permeation than larger particles Fanet al. have synthesized deoxycholic acid-conjugated chitosan, and loaded with the insulin into deoxycholic acid-modified nanoparticles (DNPs). It can overcome multiple intestinal barriers, internalized Caco-2 cells via apical sodium-dependent bile acid transporter (ASBT)-mediated endocytosis, and promoted the intracellular trafficking and basolateral release of insulin Leeet al. developed a dual ligand functionalized pluronic-based nanoparticle for oral delivery of insulin. Chitosan and zonula occludins toxin (ZOT)-derived, tight junction opening peptide were conjugated to nanoparticles to increase the intestinal permeation of loaded insulin through the paracellular pathway Many publications have proposed the potential of gold nanoparticles (GNPs) for biomedical applications. The small size and multi-valence arrangement around the gold core elevates the capacity to improve drug biodistribution and hence effectiveness and safety in vivo instability below the critical micelle concentration Microemulsion is an isotropic, transparent and thermodynamically stable system which consists of water, oil and surfactant, usually with a co-surfactant. Droplet size is normally less than 200 nm. Structurally, they are divided into three phases: water-in-oil (W/O), oil-in-water (O/W) and bicontinuous microemulsion Momohet al. and Erelet al. have developed nano-encapsulated mucin and chitosan nanoparticles in W/O microemulsion for oral insulin delivery. They showed high entrapment efficiency and stability, sustained drug release and elevated intestinal permeation Williamset al. developed an ionic liquid-based formulation for oral delivery of insulin, and the system showed high drug loading, a better access to the intestinal absorptive surface and prevented enzymatic degradation. Molecular dynamics simulation studies have shown that ionic liquids can remove water from the surface of enzymes to the same extent as polar organic solvents like acetonitrile. However, the safety issue is the major concern and the bulking care is essential particularly during handling and transport Banerjeeet al. have developed an ionic liquid-based oral formulation of insulin. This biocompatible delivery system has good long-term stability and facilitates intestinal absorption via paracellular uptake through the opening of tight junctions, results in promising insulin oral bioavailability. Thus, ionic liquids present an unprecedented and under-explored therapeutic opportunity with immense potentials for oral delivery of PPDs Ionic liquids as low melting salts with melting point <100°C, often formulated to enhance the dissolution of poorly soluble drugs, as well as to promote drug permeation through physiological barriers. In general, ionic liquids interact with various hydrophilic and hydrophobic amino acids of a protein through an intricate balance of hydrogen bonds, disulfide bonds, ionic interactions and hydrophobic effects. When mix with water or body fluid, a more complex interplay between ions occurs, which can result in formation of microemulsions or micelles Figure ).Liposomes are generally composed of one or more phospholipid membrane bilayers surrounding aqueous inner phase with sizes from 15 nm to 10 µm Figure . Lipophilic drugs are embedded in the phospholipid layers while hydrophilic molecules are encapsulated in the aqueous inner core. This nature of liposomes that can carry both water soluble and lipid soluble drugs is called amphiphilic Suzukiet al. have prepared a chondroitin sulfate-g-glycocholic acid-coated liposomes for oral exendin-4 (Ex-4) delivery. The long term pharmacodynamic effects, of daily oral exendin-4 loaded liposomes (300 μg/kg) were better than daily subcutaneous administration of Ex-4 solution (20 μg/kg) over 4 weeks Wanget al. have used bovine serum albumin (BSA) adsorbed to cationic liposomes (CLs) to form protein corona liposomes (PcCLs) for oral delivery of insulin. The results showed great intestinal permeation, led to an increase of drug oral bioavailability and hypoglycemic effect Liposomes can be divided into six types based on their size and structures as shown in Prausnitzet al. have utilized microneedle technology for oral drug delivery. They placed a 0.5-cm2 drug loaded microneedle patch onto the arms connected to a base, and called this device a luminal unfolding microneedle injector (LUMI). Once the oral administered device reached the intestine, the polymeric material holding the spring was dissolved, led to actuation that pushed the LUMI out, pressing the microneedle patches against the intestinal wall, allowing the drugs directly penetrate the intestinal epithelium. The Rani Therapeutics company has developed a related technology that deployed oral microneedles that has been carried out in a clinical trial currently, using octreotide as a model drug. Moreover, up to 0.3 mg of drug can be loaded into LUMI, which is sufficient for many potent PPDs The inherent attractiveness of microneedle-based delivery strategy demonstrates the great suitability for various PPDs delivery, even with large molecular weight Recently, it has been reported the preclinical studies of two oral microneedle devices, a poly(methacrylic acid-co-ethyl acrylate) and PEG based microneedle device for oral insulin delivery. The microneedle capsule was designed to dissolve at pH levels encountered in the small intestine. The results showed the insulin levels instantly increased and the blood glucose was reduced within 30 min, with an oral bioavailability of over 10% Abramsonet al. have developed an ingestible self-orienting millimeter-scale applicator (SOMA) that tends to position itself to engage with GIT, designed to resist external forces such as fluid flow, peristaltic motion upon reaching a stable point on the GIT wall. It then deploys milliposts fabricated from drugs directly through the intestinal mucosa while avoiding perforation. Figure An ingestible self-orienting system is a recent invented device that physically inserts a drug-loaded millipost through the GI mucosa with promising bioavailability. Inspired by the self-orienting leopard tortoise, Banerjeeet al. have fabricated an insulin loaded mucoadhesive oral patches integrated with iontophoretic circuit and surgically placed in the intestine. It was found the iontophoresis could disrupt the tight junctions of intestinal epithelium and facilitate insulin transport via paracellular pathway, without impairment of the intestinal mucosa. However, clinical proof of oral patch technology has not yet been forthcoming. However, the limiting drug loading and stability issue upon storage shall be considered Intestinal patches consist polymeric matrix embedding drugs, usually with a stabilizer. They can adhere to the intestinal wall and positioning the drugs directly to the intestinal epithelium, and meanwhile protecting the drugs from local enzymatic degradation The following are some of the drug delivery technologies that utilize combinational strategies mentioned above Figure , in orde® had been used for oral delivery of octreotide. TPE® is an oily suspension of octreotide that consists a permeation enhancer that can transiently modify the integrity of intestinal epithelium by opening the tight junction. It also consists polysorbate-80, allow to alter the thickness of intestinal mucus, thus further promote the intestinal drug uptake. Moreover, several peptides have been incorporated into TPE® including teriparatide, leuprolide, insulin and octreotide. However, a main concern in application of TPE®, the intestinal tight junction opening that cause toxicity, or the use of food emulsifiers or other excipients might initiate autoimmune disease TPE® is an oral solid dose technology can effectively increase oral absorption of a variety of low permeability PPDs. This strategy focuses on the use of medium chain fatty acid or its variants coupled with salts, resulting in greater hydrophobicity and penetration characteristics that open epithelial tight junction ® consists three major enteric coated formats. GIPET® I, is an enteric coated tablet with drug in selected weight ratios. GIPET® II, is a microemulsion form encapsulated within an enteric coated gel capsule. GIPET® III, consists of drugs with fatty acid derivatives within an enteric coated gel capsule. Currently, the Phase I and II studies have shown the safety profile of the three formats given on a repeated basis 10, have been incorporated to increase intestinal membrane fluidity and promote transcellular drug transport. Moreover, another feature of GIPET® promotes the oral bioavailability of drugs may relate to inhibition of P-gp efflux GIPETTM is a highly developed, clinically proven platform technology that enables the oral delivery of PPDs. It protects PPDs from acid hydrolysis, enzymatic degradation, and also enhances paracellular transport TM to the oral delivery of PPDs PeptelligenceThiolated mucoadhesive polymers (thiomers) that are capable of forming covalent bonds with intestinal mucus glycoproteins via thiol/disulfide exchange reactions. Thus, thiomers modified delivery system enhances the intestinal mucoadhesion, prolongs the retention in GIT and lead to higher oral bioavailability. In addition, thiomers also exhibit enzyme inhibitory, permeation enhancing and efflux pump inhibitory properties. However, thiomers are rather unstable in formulation form as they are subject of thiol oxidation at pH ≥ 5 unless sealed under inert conditions. Therefore, the use of pre-activated thiol groups might be an interesting approach to enhance its stability Melanieet al. generated and expressed functionally active colony-stimulating factor (G-CSF) as a recombinant fusion protein incorporated with Tf to evaluate the function of Tf as a carrier for oral delivery of G-CSF. The results demonstrated that the Tf moiety of the fusion protein not only promoted the drug permeation over the GI epithelium, but also protected the drug from enzymatic degradation Transferrin (Tf) is an endogenous serum protein that transports iron to cells expressing the Transferrin receptor (TfR) through TfR-mediated endocytosis. Studies have applied Tf to prepare drug carrier system to deliver PPDs, genes and poor soluble drugs to the target tissues including intestinal epithelium and blood brain barriers that abundantly express Tf receptors ®-coated capsule, and currently has completed Phase III, indicating it is a clinically advanced oral peptide format. Briefly, it uses lauroyl carnitine chloride as the permeation enhancer to promote intestinal drug permeation, and citric acid as a pH lowering agent, lowering pH to reduce protease activity, as well as encapsulating within a Eudgradit® capsule to prevent the drugs from acidic degradation in the stomach ® (Emisphere), IN-105 (Biocon) and GIPET (Merrion). What stands out about these formulations is their simplicity compared with highly complex delivery constructs Oral sCT (Ostora™) is built around coated citric acid vesicles in a EudragitOramed is a carrier system used for oral delivery insulin and GLP-1, which was developed by the Oramed Pharmaceuticals. Ormade's oral insulin is available as ORMD-0801, it allows to protect drug from enzymatic degradation and elevate the intestinal permeation of insulin. Ormades oral insulin was investigated for both type I and type II diabetes. It is currently under phase II clinical trial for oral insulin delivery and phase I trial for oral GLP-I delivery (NCT02535715) Orasome is a polymer-based liposome for oral delivery of insulin and human growth factor, which was introduced by the Endorex Corporation. This formulation allows to protect the loaded PPDs from acidic degradation in the stomach and protecting the drugs from the bile salts Q-Sphera™ technology is a novel platform to individually print narrow size distribution particles of approximate 30 μm to generate predictable pharmacokinetic profile. This micro-piezo technology was developed by the MidaTech This technology allows to solubilize potent molecules that have minimal solubility at biological pH for oral delivery Oleotec™ and Soctec™ gastro-retentive technologies were introduced by the Skyepharma. This strategy mainly focuses on promoting the drugs being absorbed in the stomach. Briefly, the technique prolongs the retention of the drugs within the stomach, and gradually releasing the encapsulated drug without being degraded by the acidic environment A variety of intestinal cell types has been identified and characterized with different surface receptors which could be potential targets for oral PPDs delivery. The therapeutic applications of most PPDs largely depend on receptor-mediated endocytosis, and the relative affinity to these receptors are crucial. Therefore, targeting these stimulating endocytosis receptors on intestinal cell surface has drawn great attention for delivery of PPDs. For this purpose, surface modified drug delivery systems or ligand-grafted drugs are required. In the following sections, the use of ligands for targeting the major receptor of different types of intestinal cells will be discussed Figure .Enterocytes are hyperpolarized epithelial cells with a columnar shape. They are the most prevalent cell type and are often targeted for the oral PPDs delivery. Several receptors have been reported to be expressing on the apical surface of enterocytes. Ligands, including vitamins, proteins, monoclonal antibody fragments and oligopeptides are often used for enterocyte targeting Liet al. used folic acid as a targeting ligand that grafted on nanoparticles to target the proton-couple folate transporter expressed on intestinal enterocytes, it mediated the endocytosis and facilitated the permeation over the intestinal mucosa Zhenget al. had developed an EGP peptide which targeted the heparan sulfate proteoglycans on the intestinal enterocytes. The EGP modified nanoparticles promoted the enterocytes uptake involving caveolae-mediated endocytosis and avoided lysosomal entrapment, thus facilitated the direct apical-to-basolateral transcytosis. Oral administrated insulin EGP NPs generated a strong hypoglycemic response on diabetic rats with 10.2-fold increase in bioavailability compared with free insulin in vivo, which is remarkably beneficial for its oral delivery Vitamins are commonly used ligands to decorate delivery systems for targeting specific intestinal cell receptors. Since they are very stable, safe with easy tunability. Vitamin B12 and biotin (vitamin B7) has been used for intestinal enterocyte targeting and showed promising results. Folic acid (vitamin B9) and thiamine have also been used as ligands for oral targeted delivery Microfold cell (M cells) are one type of intestinal epithelial cells mainly located in the epithelium of Peyer's patches. Various types of cargo can be transported across M cells, such as antigens, bacteria, viruses and particles. Without passing through lysosomes, the cargo can avoid the lysosomal degradation which is a major benefit for transcytosis of the cargo. Furthermore, the undeveloped microvilli and glycocalyx structures of M cells allow the cargo to permeate over easier compared with the enterocytes Escherichia coli-derived protein that showed GP2-targeting property. Fanet al. have developed a mucosal vaccine FimH-chitosan-pVP1 which exhibited great M cell-targeting capability, and this vaccine loaded FimH modified delivery system drastically uptakes by intestinal M cell, and promotes the dendritic cells maturation via TLR4-dependent signaling pathway Glycoprotein 2 (GP2) is a highly expressed transcytotic receptor found on the intestinal M cells Jinet al. have developed a trimethyl chitosan chloride (TMC) nanoparticle modified with CSK peptide for oral insulin delivery. The results showed the CSK peptide has significant effect on promoting drug permeation over intestinal epithelium, and the insulin loaded CSK modified nanoparticles produced a better hypoglycemic effect, with a 1.5-fold higher insulin oral bioavailability compared with drug solution Goblet cells make up to 16% of the total intestinal epithelial cells and are responsible for producing mucins Ramakrishnaet al. have successfully targeted the mannose receptor expressed on DCs using a fully human mannose receptor-specific antibody, B11, as a cargo to deliver human chorionic gonadotropin hormone. The results demonstrated B11 has great targeting capability toward DCs, and that mannose receptors and TLRs contribute towards activation and maturation of DCs by a mechanism that may be driven by a combination of peptide antigens and adjuvants Dendritic cells (DCs) play a key role in protective immunity against pathogens Enteroendocrine cells (EECs) are epithelial cells scattered throughout the whole GIT, which account for about 1% of the total intestinal cells Nagatakeet al. reported that EECs expressed a tight junction membrane protein, claudin-4 (Cld4). Orally administered luminal antigens targeting Cld4 were found to be taken up by Cld4+ cells, indicating that Cld4-mediated transport can be a potential pathway for targeting delivery of PPDs. In addition, it was found that orally administered luminal antigens were taken up by the Cld4+ EECs, raising the possibility that EECs may also play a role in initiation of mucosal immunity Shresthaet al. introduced a lipid-based nanoparticle which can act as endogenous ligands stimulating the release of GLP-1 via lipid-sensing pathways in enteroendocrine L cells Xuet al. have developed an innovative oral nanosystem to increase GLP-1 production and promote the oral absorption of peptides. The results showed the nanosystem triggered endogenous secretion of GLP-1 and increased its oral bioavailability by 4%. The nanosystem synergizes its own biological effect with the encapsulated peptide drug leading to a significant improvement of insulin resistance and glucose tolerance. This formulation strategy represents a promising approach for oral PPDs delivery in incretin-based diabetes treatment et al, the team has developed and compared different fatty acid-targeted nanocarriers and evaluated the L cell stimulation induced by the nanocarriers in vitro and in vivo. The results showed the DSPE-PEG2000 modified lipid-based nanocarriers had increased oral bioavailability of endogenous GLP-1 up to 8-fold in normoglycemic mice, and strengthened its biological effect So far, very limited studies have focused in EEC targeting in oral drug delivery. Rumioet al. further studied the various TLRs presented on Paneth cells by orally delivering TLR agonists, and the results showed that the TLR3 agonist polyinosinic-polycytidylic acid also led to Paneth cell degranulation Paneth cells usually assist in maintaining the microbiome and are located at the crypts of intestinal villi. They have a longer survival time (up to 60 days) compared with enterocytes The Peyer's patches of the small intestine play a central role in the intestinal immune system. The M cells of the Peyer's patches actively take up high-molecular weight components and are highly active in the process of phagocytosis and transcytosis. Thus, their main role is to take up luminal bacteria or large particles, transfer to DCs in the M-cell pocket for initiation of mucosal immune responses, and contribute to the homeostasis of the intestinal immune system The new generation of polymeric biomaterials, which should be adaptive, complex, and intelligent. Thus, generally cover two main characteristics. First, biocompatible materials with controllable rigidity and functionality, forming polymeric biomaterials which can be widely used in oral drug delivery, tissue engineering, etc. Second, the biomaterials often “encoded” with information which could be read by proteins on intestinal cell surface, contributing significantly to intestinal cell-cell/cell-matrix communication. This information role of biomaterials can be demonstrated not only as intestinal cell targeting reagents but also as immune adjuvants in GIT Saccharomyces cerevisiae and can be loaded with many classes of PPDs As an intestinal immune adjuvant, most carbohydrates are widely found in plants, bacteria, and yeast. They are safe, low toxic, biodegradable, with high adjuvancy, thus, the carbohydrate-based adjuvants are promising candidates Haddadiet al. conjugated α-MOS with PLGA, the result showed the delivery system modified by α-MOS could promote phenotypic and functional maturation of DCs Moreover, there are many other carbohydrate-based biomaterials, which have great intestinal bioadhesion, intestinal cell targeting properties, as well as intestinal immune modulating properties. There are many literatures reported that carbohydrates act as adjuvants through binding to specific innate immune receptors , subsequently activate macrophages, DCs, NK cells, T lymphocytes or B lymphocytes, promoting the production of immune-related molecules, such as cytokines, antibodies, etc The development of successful treatments involves not only the discovery of new therapies but also their adequate delivery to their targets. In this context, oral delivery of PPDs remains being a highly challenging endeavor. If we take into account that the first attempt to administer insulin orally was carried out in the 1920s and that, so far, there are very limited marketed oral formulations containing such large molecules, this enterprise may seem rather disappointing (Table Advanced delivery systems with novel biocompatible material and potential ligands, have demonstrated great potential in targeting different intestinal cells. However, limited numbers of receptors and ligands are available. In-depth understandings of the GIT biology in the molecular level are crucial for the discovery of new potential new receptor-ligand pairs. Based on the nature of disease and PPDs, single or combined receptor-ligand pairs could be used for intestinal cell targeting in future applications. Additionally, the cellular uptake pathways of oral drug delivery systems have not been comprehensively understood, and that poses gaps in knowledge regarding the interaction of PPDs delivery systems with the GI barriers and the dynamics PPDs metabolism. Further, as in most of the studies described herein, sustainable and tunable drug release for PPDs is still a challenge. The development of novel biocompatible materials with stimuli-responsive ability could be a potential solution. As a crucial type of biomaterial, we consider carbohydrates not only as matter or a structural component but also as information or signaling molecules. Although most of the discussed applications are still far from clinical use, carbohydrates deserve to be developed into next-generation biomaterials for oral drug delivery systems with great potential.in vitro and in vivo studies could be performed under uniform conditions to enable accurate comparisons of various approaches. Thus, the future lies in tackling these hurdles and exploiting these novel approaches for oral PPDs delivery in the clinic.Lastly, even though multiple intestinal cells targeting delivery systems showed great potentials for oral delivery of PPDs, and several formulations are currently in advanced clinical trials, and disruptive novel technologies questioning previously established ideas have been proposed Table . However


CFTR gene addition-technology to the patients’ lungs has been slow and not yet developed beyond clinical trials. Improved delivery vectors are needed to overcome the body’s defense system and ensure an efficient and consistent clinical response before gene therapy is suitable for clinical care. Cell-based therapy–which relies on functional modification of allogenic or autologous cells ex vivo, prior to transplantation into the patient–is now a therapeutic reality for various diseases. For CF, pioneering research has demonstrated proof-of-principle for allogenic transplantation of cultured human airway stem cells into mouse airways. However, applying a cell-based therapy to the human airways has distinct challenges. We review CF gene therapies using viral and non-viral delivery strategies and discuss current advances towards autologous cell-based therapies. Progress towards identification, correction, and expansion of a suitable regenerative cell, as well as refinement of pre-cell transplant lung conditioning protocols is discussed.Prognosis of patients with cystic fibrosis (CF) varies extensively despite recent advances in targeted therapies that improve CF transmembrane conductance regulator (CFTR) function. Despite being a multi-organ disease, extensive lung tissue destruction remains the major cause of morbidity and mortality. Progress towards a curative treatment strategy that implements a  CFTR) gene (Cystic fibrosis (CF) is an inherited, multi-organ disease caused by mutations in the CF transmembrane conductance regulator (TR) gene . CF is pTR) gene . The CFTTR) gene . CFTR dyTR) gene . SymptomTR) gene .CFTR mutation in the population. CFTR modulator therapies result in improved lung function and better quality of life for patients with CF . Even iin vivo delivery of CFTR to the airway cells is to correct the airway cells ex vivo followed by transplanting the corrected cells to repopulate the patient’s lung with CFTR-corrected cells . A noveiewed in .in vitro transcribed CFTR mRNA complexed with lipids to the lungs (Orphan drug MRT5005) is a therapeutic forerunner currently being tested in a phase 1/2 trial (NCT03375047) which has highly anticipated results for in vivo correction of CFTR.Exogenous nucleic acids are susceptible to degradation by nucleases and can trigger an immune response upon cellular entry . TherefoIn vivo delivery of ASOs to the airway epithelium of mice initially resulted in no significant uptake (dystrophin gene which can be treated by exon 51 skipping (reviewed in (NCT02564354). In the following phase 1b safety study with 70 enrolled participants Eluforsen was well-tolerated (NCT02532764), supporting the therapeutic translation of novel mRNA-based therapies for patients with CF. ASOs have also been tested for their ability to correct CFTR splicing mutations, which constitute 13% of disease-causing CFTR mutations, and result in no functional protein being produced . ASOs are short synthetic oligonucleotides that are chemically modified to bind to target RNA, offering a tool for direct mRNA restoration . In vivot uptake . It was t uptake . Advanceiewed in ). An ASOiewed in . In a phiewed in  . NumeroViral vector-mediated gene delivery systems take advantage of a virus’s natural ability to evade the lung mucus barrier and transduce the airway by inserting their DNA into epithelial cells. The natural tropism of viral vectors enables preferential targeting of the airway progenitor cells–the basal cells–so that multi-lineage transgene expression can be achieved . Other aIn the early 2000s, human CF clinical trials tested aerosolized viral vectors, including AAV serotype 2 (AAV2). Based on delivery of CFTR complementary DNA (cDNA) to the human nasal cavity and lung, AAV was determined safe, but failed to demonstrate significant clinical benefit in restoration of lung function . These fCFTR expression due to the transient episomal expression nature of AAV, and avoid the need for repeat dosing of the AAV vector, Cooney and colleagues designed a novel integrating AAV-based CFTR-expressing vector (termed piggyBac/AAV), which demonstrated efficient transduction and persistent expression in primary human CF airway cells in vitro and in mouse airways in vivo -CFTR to CF pig airways  have a larger packaging capacity (approximately 8 kb) which makes them more compatible for full length CFTR packaging . Additio airways . This wa airways . More re airways . In supp in vivo .Progression of viral vectors as a clinical therapy for CF remains contingent on demonstrating successful long-term transduction efficacy, the safety of delivery to the CF lung and the ability for repeat dosing . Some ofin vivo gene therapy. Exosomes have been demonstrated to deliver human CFTR mature glycoprotein, as well as CFTR mRNA, in both Chinese hamster ovarian cells  that found that repeated administration of GL67A liposome significantly, yet modestly, stabilized lung function in the treatment group (n = 62) vs. placebo (n = 54) in CF adults or children aged 12 years or older enrolled in this trial  are used to encapsulate and deliver therapeutic RNA formulations to the lung via intranasal or systemic routes. However, LNPs differ in their composition, often carrying cargo within a non-aqueous core . Recent in vivo gene therapy. This approach integrates advances in stem cell biology with the well-established platform of in vitro gene delivery and repair strategies. The exemplar for cell-based therapy is hematopoietic stem cell transplantation, which is successfully performed in over 50,0000 patients per year world-wide , has been demonstrated in a clinical trial for hematological malignancies  identification, 2) CFTR correction (addition or editing), 3) expansion, and 4) transplantation back into the patient’s lungs . The remOne of the obstacles to cell-based therapy for CF is the identification of an optimal self-renewing cell that can also differentiate into the cells of the airway epithelium. In this review we discuss therapeutic perspectives of using adult tissue-resident basal stem cells, yet first we will briefly describe advances in the field using mesenchymal and induced pluripotent stem cells (iPSCs).in vitro and in vivo studies are needed before this approach is considered viable. Until then, there remains uncertainty surrounding the capacity of MSCs to effectively restore CFTR function in the respiratory epithelium.Mesenchymal stem cells (MSCs) are an allogenic source of cells that have been investigated for their application in CF cell-based therapy due to their immunomodulatory and anti-inflammatory properties (reviewed in ). In favin vitro  or 2-basal stem cells. The strengths and limitations of both these will be discussed here briefly. Firstly, several reports have demonstrated successful directed differentiation of human iPSCs into AECs in vitro . The iPSin vitro . As suchin vitro to differentiate into a pseudostratified airway epithelium exhibiting CFTR activity  . These iactivity . Howevernterface . FurtherTo improve the chances of successful cell transplantation to the lung, it is possible that tissue-resident adult stem cell from the lung itself would be a better suited candidate. For some time, there had been a lack of clarity as to the identity of airway stem cells. However, increased knowledge of stem cell biology together with the characterization of lung progenitor lineage has brought emerging consensus that basal cells are a stem cell or progenitor cell of the airway epithelium . ImportaThe existence of multiple progenitors with various differentiation capacities has been detected in human bronchial xenografts . This isin vitro in airway epithelial cells can be achieved by direct addition of genetic material as discussed in the sections above . An alternative to introducing new material is to correct the defective CFTR via gene editing.Correction of CFTR in vivo application of CRISPR/Cas9 was employed for a person with a mutation in the CEP290 gene which causes retinal degeneration –Cas-associated nucleases, programmable nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). The first neration . Clinicaneration . In the CFTR sequence to robustly restore CFTR function in human intestinal stem cell organoids derived from patients homozygous for the Phe508del mutation . The coin vitro  is one ain vitro  and mainin vitro . CF and in vitro . Importain vitro . Yet, fuactivity  and diffactivity . If expain vitro expansion of basal cells often have decreased CFTR ion transport function . Can baMoreover, challenges will likely be met in transitioning gene- and cell-based therapies to clinical care. Discussions of the difficulties in developing and sustaining a successful business model for cell-based therapies, and the changes in clinical care necessary to make this potentially transformative therapeutic approach accessible to patients, are ongoing . As we c


To assess the time course changes in corneal topographic parameters during the one-year follow-up after Descemet membrane endothelial keratoplasty (DMEK) surgery.  Twenty-one patients (24 eyes) who underwent DMEK surgery were evaluated. Best corrected visual acuity (BCVA), endothelial cell count (ECC), central corneal thickness (CCT), mean keratometry (MK), mean astigmatism (MA), astigmatism asymmetry (AA), and higher-order aberration (HOA) were assessed at baseline and 1, 3, 6 and 12 months after the surgery using CASIA2 anterior segment swept-source OCT .  In patients who underwent DMEK surgery, BCVA improved gradually at the subsequent visits during the 12-month follow-up. A significant reduction in ECC and CCT at the 1st month was noted, which remained stable until the 6th month postoperatively. Anterior and total MK values remained unchanged, whereas changes in posterior keratometry were noticeable until the 6th month after surgery. A significant reduction in the anterior, posterior, and total astigmatism magnitude as well as astigmatism asymmetry was observed during the first 6 months after surgery. A gradual anterior, posterior, and total HOA decrease was documented until the 12th month after surgery. Negative correlations between baseline values of CCT, MK, MA, AA, and HOA and postoperative variations in those parameters at consecutive follow-up time points were observed. Accordingly, negative correlations between baseline CCT and postoperative changes in corneal topographic parameters after surgery were found.  The stabilization of most corneal topographic parameters takes place within 6 months after the procedure, whereas HOA reduction and BCVA improvement gradually occur during the first year after surgery. Preoperative values of corneal topographic parameters strongly determine their changes detected after DMEK surgery, which may suggest that early therapeutic intervention results in better visual outcomes. In the last few years, Fuchs corneal endothelial dystrophy and pseudophakic bullous keratopathy have become the most common indications for corneal transplantation . Less thSince the introduction of the DMEK technique, changes in endothelial cell count (ECC), best corrected visual acuity (BCVA), and central corneal thickness (CCT) after surgery have been widely studied , 10–13. In the present study, we aimed to assess the time course changes in corneal topographic parameters during a one-year follow-up after DMEK surgery. We also explored the relationship between dynamic variations in corneal curvature, CCT, BCVA, and ECC between individual visits throughout the 12-month postoperative observation time. To the best of our knowledge, this might be the first retrospective study to provide such a broad and precise analysis of dynamic variations in corneal keratometry, astigmatism, astigmatism asymmetry, and HOA values based on anterior segment swept-source optical coherence tomography (SS-OCT) recordings.This retrospective case series included 24 eyes after DMEK surgery, which was carried out due to various causes of endothelial decompensation: Fuchs endothelial corneal dystrophy (FECD) or pseudophakic bullous keratopathy (PBK). The patients enrolled in the study were operated on at the First Ophthalmology Clinic in Szczecin in 2018–2020 and then monitored 1, 3, 6, and 12 months after the surgery. All participants underwent a complete ophthalmologic examination, including the following: best corrected distance visual acuity with Snellen charts, slit lamp biomicroscopy, and a detailed fundus examination after pupil mydriasis. Intraocular pressure (IOP) measurement and corneal quality parameters were evaluated with swept-source anterior segment optical coherence tomography (AS-OCT) at each follow-up visit.2.Donor corneas were obtained with the multiorgan procurement method and in the dissecting room during autopsy. Corneoscleral buttons were stored in Eusol-C medium  in hypothermic storage at 2–6°C at the West Pomeranian Eye Tissue Bank in Szczecin. The prestorage evaluation of the endothelium was performed by specular microscopy . All corneas had an endothelial cell count of at least 2800 cells/mmDirect preparation of the graft before transplantation took place in the operating theatre. All grafts were stripped and left on their natural support immersed in 0.06% trypan blue dye . Then, grafts were trephined by the surgeon to the desired diameter (6.0–8.0 mm) using a Hessburg-Barron donor corneal punch .Each surgery was performed by the same surgeon. All patients underwent prophylactic basal laser iridectomy at 6 o'clock position before endothelial keratoplasty to minimize the risk of postoperative pupillary block. Patients with retinal diseases significantly affecting visual acuity were excluded from the study. All procedures were performed with peribulbar block.The epithelium of the recipient was marked to guide the subsequent Descemetorhexis and to allow the correct positioning and perfect centring of the transplanted donor flap. The anterior chamber (AC) of the eye was then entered through a clear corneal incision. After the injection of a hyaluronate viscoelastic material, the endothelium and the Descemet membrane were stripped using an inverted Price-Sinskey hook. The stripping diameter was 1 mm wider than the graft diameter. The viscoelastic material was rinsed after stripping. The removed flap was exposed on the anterior surface of the receiver's cornea to verify its integrity.The surgery was performed following the “no-touch” technique. The trephined DMEK graft was carefully detached from the surrounding Descemet membrane, immersed in sterile balanced salt solution, and aspirated into the transparent glass cartridge . The rolled graft was injected into the AC with slow and continuous pressure through the main incision (2.4 mm). The graft was then unfolded and positioned using the tap-tap technique. After ensuring the correct orientation and centration, the graft was pressed against the recipient stroma by injecting SF6 underneath. One nonabsorbable 10-0 suture was applied to the operating port and kept until the first-week follow-up visit.All patients were instructed to stay in a supine position until the first postoperative flap position control was done. In the case of a pupillary block or ocular hypertension, topical mydriatics were administered, or if this procedure was insufficient, a small quantity of air was released from the AC in the operating theatre. The postoperative treatment consisted of a topical antibiotic given 4 times a day for 1 week and topical preservative-free dexamethasone sodium phosphate 8 times a day for the first month. The topical steroid was tapered down to one drop every other day and then discontinued over a 1-year period.Visual function was assessed in all participants by evaluating BCVA using a Snellen chart. The result was recorded in the decimal system.The ECC was measured at each follow-up visit using a specular microscope .Both corneal thickness and keratometry values were determined using a swept-source anterior segment OCT CASIA2 . During the entire observation period, the CASIA2 was placed in the same room under the same lighting conditions. All measurements were taken by trained operators. Operators gently held patients' eyelids to avoid putting pressure to the globe. The scan was performed using the autoalignment function. The CASIA 2 measurements were obtained with the corneal map mode of the anterior segment module. The images were analyzed by built-in 2D analysis software that automatically calculated the measurements along with the structural outlines and reference lines. The outline tracer was edited where needed.μm), mean keratometry values (D), astigmatism power (D) and axis (°), astigmatism asymmetry (D), and higher-order aberration (HOA) power (D) were recorded and analyzed at assumed time points after surgery using the Fourier analysis 3D/2D function. Measurements were read from both the anterior and posterior surfaces of the cornea, and the total values were taken into account. All parameters were assessed at optical zones (OZs) with 3 and 6 mm diameter. The image quality was assessed during acquisition by the operator. Only well centred measurements with high-quality indexes were included in the study.Central corneal thickness (CCT) . A n = 24). No graft failures or rejections were observed. The preoperative characteristics of the patients, as well as donor graft characteristics, are shown in μm, respectively.Twenty-four eyes of 21 patients qualified for DMEK surgery . No significant changes in ECC values (cells/mm2) at the 1st, 3rd, and 6th month after surgery were noted. Subsequently, we observed a significant reduction in ECC at the 12th month compared to the values recorded at the 6th month.Next, the dynamics of the changes in endothelial cell count after DMEK surgery were analyzed . A signiμm)  , CCT decp=0.38 and p=0.26, respectively).Further, we analyzed the dynamics of changes in keratometry recordings after DMEK surgery . No signAfterwards, the influence of the DMEK procedure on astigmatism changes was evaluated . We obseNext, we evaluated irregular corneal astigmatism with an asymmetry of the astigmatic components . We obseTo provide a broad-based assessment of corneal topography after DMEK surgery, we analyzed the higher-order corneal aberrations (D) that may influence vision quality and acuity . A graduIn the next step, we evaluated the potential relationships between corneal parameters at consecutive time points after surgery. We detected that preoperative values of corneal parameters strongly determined their changes detected after DMEK surgery. Accordingly, we found negative correlations between baseline values of CCT, keratometry, astigmatism, astigmatism asymmetry, and HOA and postoperative variations in those parameters at consecutive follow-up time points . This fiImportantly, we determined that baseline CCT strongly influences the changes in other corneal topographic parameters after surgery. Endothelial corneal transplantation techniques are constantly being improved, and their continuous development contributes to more effective and safer treatment of patients with endothelial damage.Many previous studies have focused on the variations in visual acuity and corneal topographic parameters in 6- or 12-month observation periods, comparing them to the baseline conditions , 23, 24.This retrospective study provided such detailed analysis of the relationships between the dynamic status of corneal topographic parameters, corneal thickness, best corrected visual acuity, and endothelial cell count analyzed at several postoperative follow-up points throughout a 1-year observation period.Our data provided evidence that posterior keratometry recordings decreased just after the operation at the 1st month postoperatively compared to those recorded preoperatively, which indicates a steepening of the posterior corneal curvature. Altogether, the potential explanation is that the recipient endothelium is replaced with an undersized donor graft; thus, the peripheral margin of the stripped area is deprived of endothelial cells, causing marginal thickening that is due to the unsealed endothelial cell barrier at the peripheral corneal area. This consequence occurs with subsequent steeping of the posterior corneal surface. With time, the donor cells migrate and fill the gaps between recipient and donor tissue, thus leading to a resolution of the peripheral oedema and a flattening of the posterior corneal surface. Subsequently, the cornea returns to a physiologically hydrated status. Indeed, we found a significant increase in posterior keratometry values at 3 and 6 months after DMEK surgery, with subsequent stabilization at 6 to 12 months. Van Dijk et al. in their large prospective study on DMEK indicated that a potential cause of posterior keratometry decrease is the specific corneal healing process. In the early phase after DMEK surgery, the cornea shows central thinning while the periphery is still edematous, creating a steepening of the posterior cornea curvature and a flattening of the anterior cornea curvature, which results in a “hyperopic shift.” As the transplanted cornea returns to a physiological hydration status, the induced hyperopic shift is again reduced but still detectable in comparison to the preoperative power [In our study posterior keratometry outcomes coincided with a decrease in ECC at the 12-month compared to the 6-month measurements since endothelial cells have limited potential to proliferate. We cannot exclude the possibility that a decrease in ECC results in an increase of CCT at the 12th month postoperatively since we documented an increase in CCT at the 12-month compared to the 6-month values.Indeed, similar patterns of ECC and CCT changes were documented in previous studies , 23–25.μm might have a thicker cornea at the 12-month follow-up. This finding is in line with our observation that baseline CCT determines the variations in corneal thickness after surgery since we found a strong negative correlation between baseline and postoperative changes in corneal thickness at subsequent postoperative visits.Importantly, Brockmann et al., in their prospective observational study , noted tPA parameter, which was calculated as the posterior to anterior corneal curvature radii ratio, as surrogate markers to identify eyes that might be at risk of a greater postoperative hyperopic shift after DMEK [Furthermore, we observed that the DMEK procedure did not impact anterior or total keratometry values. This observation is in line with the data of Kwon et al., who documented that total keratometry did not change significantly after the DMEK procedure and that postoperative values were comparable to those in the healthy cohort. Accordingly, the authors found that the anterior corneal surface remained relatively unchanged, whereas the posterior corneal surface displaced forward . On the ter DMEK . Bearingter DMEK .Taken together, the observed inconsistences between studies might have been associated with the use of different topography devices, possible differences in operating technique and donor tissue preparation, different conditions of donor graft storage, or differences in the criteria for study group selection. Thus, we suggest that while carrying out such procedures, surgeons should create their own intraocular power nomograms for cataract surgery before or during DMEK surgery based on their definite observations and own experience.Data on corneal aberrations after DMEK surgery differ between studies and are not consistent. In terms of astigmatism power astigmatism power, we documented a reduction in the total astigmatism power detected exclusively between the 1st and 3rd months after the procedure with subsequent stabilization of astigmatism magnitude. Contrary to our study, the recent report by Gundlach et al. presented the opposite pattern of a decrease in astigmatism from the 3rd to the 12th month postoperatively . HoweverImportantly, we detected that preoperative values of corneal topographic parameters strongly determined the changes detected after DMEK surgery since we found negative correlations between their baseline values and postoperative variations. This result strongly corroborates the notion that early therapeutic intervention results in better visual outcomes.In conclusion, the results of our study provide valuable information regarding the dynamics of postoperative changes in corneal parameters after the DMEK procedure. The presented data clearly demonstrate that the stabilization of most corneal topographic parameters  takes place within 6 months after the procedure, whereas HOA and BCVA gradually improve during the first year after surgery.


Patient-derived cancer organoids have taken a prominent role in pre-clinical and translational research and have been generated for most common solid tumors. Cancer organoids have been shown to retain key genetic and phenotypic characteristics of their tissue of origin, tumor subtype and maintain intratumoral heterogeneity and therefore have the potential to be used as predictors for individualized treatment response. In this review, we highlight studies that have used cancer organoids to compare the efficacy of standard-of-care and targeted combination treatments with clinical patient response. Furthermore, we review studies using cancer organoids to identify new anti-cancer treatments using drug screening. Finally, we discuss the current limitations and improvements needed to understand the full potential of cancer organoids as avatars for clinical management of cancer therapy. Cancer remains one of the leading causes of death in the 21st century. Despite enormous progress in the identification of mechanisms of tumor progression and treatment resistance and the development of new tumor targeted treatments many patients do not get cured. The main challenge remains to achieve accurate patient selection. Tumor biopsies used for clinical diagnostics do not capture the extensive intratumoral heterogeneity that masks emergent tumor clones with intrinsic or acquired resistance. This can result in patients receiving suboptimal treatment, or overtreatment leading to long-lasting harmful side-effects. The development of specific biomarkers and predictive model systems are therefore essential to personalized treatment leading to more durable responses with fewer side effects.in vivo drug metabolism which are currently not accounted for in cancer organoids  up to October 2020. For the purpose of this review, cancer organoids were defined as stem-cell derived three-dimensional cell culture models derived from primary patient-derived solid tumors using a basement membrane extract (BME). Articles describing patient-derived-xenograft (PDX)-derived cancer organoids, iPSC based models or three-dimensional cell culture systems lacking a BME (i.e. slice cultures or tumor-on-a-chip approaches) were excluded. Search keywords included [ORGANOID] or [TUMOROID] and, [CANCER] and [TREATMENT]. Only articles in which cancer organoids were subjected to treatment and correlated to patient outcome or potential biomarkers were included. Establishment, characterization and comparison of cancer organoids towards the parental tumor have already been previously described and fall outside the scope of this review , 7.A total of 60 studies were included in this review. For almost all papers the cancer organoids used were (previously) compared on a genetic and phenotypic level to the parental tumor in order to validate the model. Study characteristics are summarized in In 2015 the first colorectal cancer (CRC) organoid biobank was established and used for drug screening. Based on these drug screens, gene-drug interactions could be studied to identify potential biomarkers and study the molecular basis for drug response to both new therapeutic agents as well as current standard-of-care chemotherapeutics . All but one of the patients used for organoid derivation showed poor clinical response to chemotherapy which was recapitulated in the corresponding cancer organoids, though organoids from the patient that showed clinical response were not available for drug testing . This ovA similar approach was taken using gastric cancer organoids. In one study, gastric cancer organoids derived from one patient at pre-treatment were sensitive to standard-of-care chemotherapy (5-fluorouracil (5-FU), cisplatin, oxaliplatin and irinotecan) which reflected the complete pathologic response in the patient after chemoradiation, albeit the contribution of radiotherapy to the clinical response was not investigated . In anotAscites-derived gastric cancer organoids showed a heterogeneous response towards standard-of-care chemotherapeutics  between patients, similar to the mixed clinical responses seen in patients with peritoneal metastases, though no direct clinical comparison could be made .CRC organoids were used to identify patients that benefit from the PARP inhibitor olaparib and cross sensitivity to oxaliplatin, which causes PARP-dependent DNA damage repair. In two patients that responded to oxaliplatin the organoids were sensitive to both olaparib and oxaliplatin. In another patient that responded, the organoids showed resistance to both treatments. Notably the organoids from this patient were highly responsive towards panitumumab which was also part of the clinical treatment and might have been a main factor in the clinical response and explain the discrepancy between organoid and clinical response .The TUMOROID study used organoids derived from metastatic CRC and correlated the organoid treatment response towards the corresponding clinical response. Organoids were able to predict the response to irinotecan-based therapies in more than 80% of patients without misclassifying patients who responded to the treatment. This predictive value was not identified for 5-FU and oxaliplatin combined treatment. A possible explanation for this could be that the tumor micro-environment , not present in organoids, might influence the efficacy of one treatment more than the other . In anotin vitro. Furthermore, two treatment-refractory patients were treated with a drug that was selected based on the drug sensitivity observed in their corresponding organoids. One patient received vandetanib (pan-tyrosine-kinase inhibitor) which strongly reduced organoid viability, however no clinical response was observed. For another patient, gemcitabine showed an initial partial clinical response but after two additional months of treatment, disease progression was again observed , and reflected clinical resistance in a patient who, based on molecular markers (EGFR amplification and KRAS wild-type), was expected to respond to the treatment .Rectal cancer organoids were exposed to standard-of-care chemotherapy ) or radiotherapy . A high correlation (r=0.86) for 5-FU or FOLFOX was observed when compared to the progression-free survival (PFS) of the corresponding seven patients. For radiotherapy, organoids that showed resistance to radiotherapy were derived from previously irradiated tumors or from tumors that showed no to minimal clinical response. On the other hand, more radiosensitive organoids were derived from patients who had a minimal 50% reduction in tumor circumference endoscopically or a near-complete or a clinical complete response following radiotherapy . AdditioAs a proof-of-concept, the influence of KRAS mutation-mediated resistance to EGFR-targeted therapy using cetuximab was tested in rectal cancer organoids. In accordance with clinical trial data, KRAS-mutated organoids showed more resistance towards cetuximab compared to the KRAS wild-type organoids . These fMultiple studies have used organoids to screen for efficacious targeted agents based on genomic targetable variants present in the organoids. This approach was taken using gastric cancer organoid biobanks, CRC cancer organoids and in esophageal cancer organoids in which drug screening approaches identified patient subsets with potential vulnerability to new targeted agents , 24, 67.Finally, organoids were used to improve current treatments. CRC organoids showed an enhanced response to radiotherapy when organoids were simultaneously exposed to radiosensitizer APG-880 . PeritonPancreatic cancer is one of the most lethal malignancies as it is often diagnosed in an advanced stage, has a high recurrence rate and only a minor survival benefit can be achieved with systemic therapy. This resistance to gemcitabine was also observed in pancreatic cancer organoids, though no clinical correlation could be made .An organoid-derived pharmaco-transcriptomic signature was developed to predict drug sensitivity to gemcitabine monotherapy but it did not predict response in patients receiving a combination treatment with other chemotherapeutics . SimilarPancreatic cancer organoid cultures derived from multiple different metastatic sites from the same patient showed a differential sensitivity towards 5-FU, but not towards the other chemotherapeutics tested. This suggests the existence of cancer subclones that differ between metastatic sites which are maintained in their respective cancer organoids .Optical metabolic imaging (OMI) was used to assess treatment response in pancreatic cancer organoids and classify patients. OMI is a high-resolution fluorescence microscopy technique that quantifies the metabolic state of individual cells. This technique measures drug response in heterogeneous 3D populations faster than more traditional methods, as changes in cell metabolism precede changes in cell viability . Using ONovel approaches for pancreatic cancer organoids include the co-culture with cancer-associated fibroblasts (CAFs) and using matched pancreatic ductal organoids. CAFs were shown to increase treatment resistance which shows the importance of tumor micro-environmental aspects on treatment efficacy . Using mLiver cancer organoids can be derived from liver cancer subtypes (hepatocellular carcinoma (HCC) cholangiocarcinoma (CC) or mixed type (HCC/CC)). Overall differential sensitivity was found between organoids from different patients to chemotherapies (gemcitabine) and targeted therapies  , though Liver cancer organoids (n=7) were also subjected to targeted therapies . Sensitivity to these treatments however did not fully correlate with the presence of these driver mutations . In a moCancer organoids were also derived from pediatric hepatoblastoma. For one patient drug testing was performed on the matched normal liver and tumor organoids. This screen identified one drug (JQ1) to have an increased efficacy on tumor organoids compared to normal organoids whereas standard-of-care cisplatin had not differential effect . MatchedBladder cancer organoids were tested for sensitivity to chemotherapeutics , though no correlations could be made with patient response . Anotherin vivo and is maintained in cancer organoids. Furthermore, high-throughput drug screens in Wilms tumor organoids identified MEK and HDAC inhibitors as novel candidate interventions. Importantly, matched normal kidney organoids used in this study as well were equally sensitivity to romidepsin (HDAC inhibitor) and MEK inhibition compared to the tumor organoids. For pabinostat (pan-HDAC inhibitor) a significant increased sensitivity was observed in the tumor organoids compared to the normal kidney organoids making this the most interesting for clinical use. Using this approach, this study sets an example of using matched healthy and tumor organoids to identify treatments with the best therapeutic ratio, considering both tumor efficacy and normal tissue toxicity (A biobank (>50 organoid lines) of pediatric kidney cancer organoids was used to test treatment sensitivity towards standard-of-care chemotherapy (neoadjuvant actinomycin D (ACT-D) and vincristine; adjuvant doxorubicin and/or etoposide) on a specific subset of pediatric kidney cancers (Wilms tumor). Organoids derived from patients that received neoadjuvant chemotherapy were less sensitive to vincristine than those from patients not receiving prior chemotherapy. This suggests that resistance already develops Prostate cancer organoids were developed from neuro-endocrine prostate cancer (NEPC) and used for screening of cytotoxic drugs and identified alisertib (aurora A-kinase inhibitor). Organoids from two patients enrolled in a phase 2 clinical trial of alisertib mimicked the clinical response of the patients , supporting the potential clinical relevance of NEPC organoids as a predictive platform , 70.Castrate-resistant prostate cancer (CRPC)-derived organoid lines (n=7) could successfully be established from biopsies and circulating tumor cells from all subtypes. A very limited drug study showed a response towards androgen suppression therapy  in an organoid with androgen receptor amplification. This organoid line also showed a response to everolimus (mTOR inhibitor) and buparlisib (PI3K inhibitor) correlating with mutations in PTEN and PIK3R1 .Endometrial cancer organoids (EC-O) representing early hyperplastic endometrium (n=13) and different stages and grades were derived (n=16) and exposed to standard-of-care treatment  and everolimus. These studies showed differential response between organoids from different patients . SimilarOvarian cancer organoids (OC-O) were successfully developed from multiple stages and subtypes (56 organoid lines). Standard-of-care drugs (platinum/taxanes) as well as targeted agents (PIK3K/AKT/mTOR inhibitors or PARP inhibitors) were used to test treatment sensitivity of OC-O. Unsupervised hierarchical clustering based upon platinum/taxane sensitivity could distinguish chemosensitive high-grade serous (HGS) organoids and chemoresistant non-HGS organoids which corresponded to the clinical findings. For one patient, HGS organoids clustered with the resistant group, corresponding to its clinically chemo-resistant phenotype and status as recurrent disease. The pre-treated counterpart of this patient did cluster with the chemo-sensitive group, correlating with clinical behavior . AnotherAs a proof-of-concept, organoids from one GBM patient, progressive after standard-of-care treatment, was used to identify potential drug candidates based on genomic alterations. This identified everolimus to be a potential therapeutic agent which correlated with a partial clinical response in this case-study . Cancer Chordoma organoids, a rare spinal cancer, were established that retained PD1 positive CD8 T-cells and were used to predict response towards nivolumab (PD-L1 blockade). A dose-dependent effect was observed in both PD-L1 positive and PD-L1 negative patients which corresponds with previous findings that low expression of PD-L1 can still lead to responses towards PD-L1 blockade . This study shows that this treatment response can potentially be predicted in cancer organoids, regardless of PD-L1 status though no correlation towards individual patient response could be made .Multiple organoid lines of retinoblastoma were developed and tested for response to standard-of-care chemotherapy  which showed a similar response towards tumor cells in advanced disease in clinical practice, but here no direct comparison to the patient response was made .in vitro response to tamoxifen (estrogen receptor antagonist) matched that of the patients, showing their potential as treatment predictors. This biobank was used for high-throughput drug screening in which most, but not all, organoids responded to treatment as predicted from their mutations. Some organoid lines were insensitive to HER2 targeting despite HER2 overexpression which emphasizes the value of functional in vitro drug tests using cancer organoids  breast cancer organoid biobank was established. For 12 patients with metastatic breast cancer a comparison with patient outcome was made. In this subset, the rganoids .In another proof-of-concept study breast cancer organoids were derived from one patient and drug screening identified fulvestrant (estrogen receptor antagonist) as the most optimal treatment for this patient whereas based on genetic analysis (PTEN mutant), everolimus was expected to be the most effective treatment. Possibly this discrepancy can be explained by subclonal PTEN alterations resulting in differential efficacy of everolimus. Since this patient was not treated with either of these agents no correlation to the clinical response could be made, however it does show the additive value of drug screen on cancer organoids to genetic analysis of the tumor .Lung cancer is the leading cause of cancer mortality and can be subdivided in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The first lung cancer organoid biobank was established using 80 lung cancer (SCLC and NSCLC) patients. Drug sensitivity testing was performed for both cytotoxic drugs (docetaxel) and targeted agents (olaparib and erlotinib). Sensitivity to PARP inhibitor olaparib correlated with BRCA2 mutation status as expected. EGFR-targeting by erlotinib did correlate with EGFR mutation status in most, but not all, lung cancer organoids. In one patient that harbored an EGFR mutation but was resistant to erlotinib, a MET amplification was present explaining resistance . In anotin vitro resistance towards either one of the treatments could be confirmed in in vivo mouse models (Head-and-neck squamous cell carcinoma (HNSCC) organoids were developed and tested for treatment response towards standard-of-care chemotherapy in the metastasized setting (cisplatin and docetaxel). This study observed the highest IC50 value for docetaxel in the organoids derived from a pre-treated relapsed patient. Furthermore, e models . HNSCC oe models . Anothere models .In the past decade, cancer organoids have been established for large set of solid tumors and extensively characterized on a genetic, transcriptomic and phenotypic level. Overall, the conclusion is that cancer organoids are genetically and phenotypically stable replicates of the tissue and tumor subtype characteristics. Multiple studies have also used cancer organoids for drug screening approaches. However, only few studies have been able to make a quantitative clinical comparison to derive predictive values , 25, 51.Several limitations currently exist that need to be addressed before cancer organoids can be implemented as a co-clinical track to aid clinical decision making. First, the derivation of organoids is not equally effective for all solid cancers. For example, NSCLC has shown a low establishment rate due to frequent overgrowth of lung cancer organoids by normal airway cells . This ovFinally, the usage of exogenous growth factors and animal-derived basement membrane extracts (BME) in organoid cultures might influence reproducibility between organoid studies. There is currently no consensus on which medium components should be used, also not within one tumor type, which could explain differences found between studies with regards to treatment sensitivity. Moreover, the BMEs that are mostly used (i.e. Matrigel) feature variable compositions and the protein composition does not always reflect the stromal composition of the tissue the cancer cells were derived from. Utilizing a well-defined 3D matrix, adjusted to specific tumor types, might help to further improve cancer organoid studies .Cells from the tumor micro-environment, such as stromal cells, immune cells and endothelial cells are lacking from cancer organoids. This may limit the utility of patient cancer organoids as predictors of treatment response as the tumor micro-environment is a key determinant of therapeutic outcome and a potential therapeutic target , 76. ImpSeveral attempts have already been made towards incorporating aspects of the tumor micro-environment into the cancer organoid system . These iin vitro differentiated iPSCs towards endothelial cells were generated  in treatment experiments using cancer organoids. These differences in drug dose could lead to survival of cell populations in vitro that do not die in vivo or vice versa. Furthermore, since all studies use different drugs schedules and dosages, discrepancies of the predictive value of organoids between different studies can occur. Changes in drug concentration due to differences in diffusion of metabolites, in vivo drug metabolism and limited drug penetrance as a result of physiological barriers such as the blood-brain-barrier (BBB) can change the efficacy of a treatment option due to inadequate drug concentration in the cancer organoid. Microfluidics and an artificial BBB using organ on a chip technology may advance the field in this respect .Finally, cancer organoids can act as living surrogates (patient avatars) to use for high-throughput drug screening approaches to aid directly in the treatment of the specific patient that the organoid has been derived from and for the discovery of novel drug and targets. This possibility opens up major possibilities in the selection of personalized treatment options and the prevention of normal tissue toxicity.MVe: conceptualization, data curation, investigation, writing , and final approval. AH: analysis and interpretation of data, writing (review), and final approval. DD: analysis and interpretation of data, writing (review), and final approval. MVo: conceptualization, data curation, investigation, supervision, writing , and final approval. All authors contributed to the article and approved the submitted version.KWF Kankerbestrijding (Dutch Cancer Society) Unique High Risk (16698/2018-1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We describe a middle-age male with a past medical history of second-degree atrioventricular block type II status post permanent pacemaker placement the day prior who presented to the emergency department complaining of chest pain. Electrocardiography showed a non-paced ventricular rhythm. Chest radiograph showed the ventricular pacemaker lead located distally overlying the right ventricle apical area. On further investigation, chest computed tomography showed a perforation of the ventricular wall by the pacemaker lead prompting urgent intervention by the cardiothoracic surgery team for lead replacement and right ventricular repair.Our case illustrates the importance of timely recognition of a perforated pacemaker lead in a patient presenting with chest pain after device implantation. We additionally describe the risk factors for ventricular perforation, initial clinical presentation, and management approach. A 52-year-old man with second-degree type II atrioventricular (AV) block and recent dual-chamber pacemaker placement presented to the emergency department with left sided chest pain for the prior two hours. Electrocardiogram showed a second-degree AV block with a ventricular rate of 45 beats per minute. Pacemaker interrogation revealed atrial sensing without ventricular capture. His chest pain was exacerbated with electrical stimulation despite lack of capture and resolved after disabling the pacemaker. Further imaging (chest radiograph [CXR] and point-of-care echocardiogram) revealed ventricular pacer lead mispositioned and a possible cause of acute chest pain , 2. CoroPacemaker lead ventricular perforation is a rare event that occurs in 0.4–5.2% of pacemaker lead placements.What do we already know about this clinical entity?Pacemaker lead ventricular perforation is a rare complication of pacemaker placement and can present with chest pain, dyspnea, dizziness, syncope, or pacemaker failure.What is the major impact of the image(s)?Chest radiography followed by point of care echocardiography or computed tomography of the chest are readily available imaging studies to support the diagnosis.How might this improve emergency medicine practice?Emergency physicians should have a high index of suspicion for pacemaker lead perforation in the clinical setting of chest pain and recent pacemaker placement.Chest pain, dyspnea, dizziness, syncope, and pacing or sensing pacemaker failure are commonly encountered clinical scenarios. Point-of-care echocardiogram allows for the clinician to promptly rule out emergent conditions such as hemorrhagic pericardial tamponade. Additional workup includes CXR and echocardiogram to evaluate for pericardial effusion; however, a non-contrast CT is usually required to confirm the diagnosis. Management is targeted based on patient’s clinical stability and the extent of injuries to nearby structures.