Recent advances in the engineering of nanosized active pharmaceutical ingredients: Promises and challenges

The advances in the field of nanotechnology have revolutionized the field of delivery of poorly soluble active pharmaceutical ingredients (APIs). Nanosized formulations have been extensively investigated to achieve a rapid dissolution and therefore pharmacokinetic properties similar to those observed in solutions. The present review outlines the recent advances, promises and challenges of the engineering nanosized APIs. The principles, merits, demerits and applications of the current ‘bottom-up’ and ‘top-down’ technologies by which the state of the art nanosized APIs can be produced were described. Although the number of research reports on the nanoparticle engineering topic has been growing in the last decade, the challenge is to take numerous research outcomes and convert them into strategies for the development of marketable products

Evaluation of Microencapsulation of The UFV-AREG1 Bacteriophage in Alginate-Ca Microcapsules using Microfluidic Devices

The indiscriminate use of antibiotics and the emergence of resistant microorganisms have become a major challenge for the food industry. The purpose of this work was to microencapsulate the bacteriophage UFV-AREG1 in a calcium alginate matrix using microfluidic devices and to study the viability and efficiency of retention. The microcapsules were added to gel of propylene glycol for use as an antimicrobial in the food industry. The technique showed the number of the phage encapsulation, yielding drops with an average 100-250 $\mu$m of diameter, 82.1 $\pm$ 2% retention efficiency and stability in the gel matrix for 21 days. The gel added to the microencapsulated phage showed efficiency (not detectable on the surface) in reducing bacterial contamination on the surface at a similar level to antimicrobial chemicals (alcohol 70%). Therefore, it was possible to microencapsulate bacteriophages in alginate-Ca and apply the microcapsules in gels for use as sanitizers in the food industry.Comment: 8 pages, 5 figure

산업공정 리그닌을 이용한 약물 방출 조절형 나노입자 합성

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 산림과학부(환경재료과학전공), 2021. 2. 최인규.There has been growing interest in lignin as a promising source for drug delivery system preparation in recent years. The lignin-based nanoparticle has the potential to be preferably used because of its controllable intracellular uptake and stimuli-controlled drug-releasing ability. However, few research investigated the effect of structural characteristics of lignin on the synthesis of nanoparticles and its essential features such as size, colloidal stability, and biocompatibility. This research focuses on how the molecular structures of technical lignin (kraft and alkaline/soda lignin) affect the lignin nanoparticle properties. Based on these results, drug-encapsulated lignin nanoparticles were synthesized, and those drug-releasing efficiencies were investigated through evaluation of in vitro and in vivo biocompatibility. First, lignin fractions with different molecular weights (Mw 1,460 ~ 12,900), phenolic hydroxyl group (total 3.7 ~ 6.2 mmol g-1), and main internal linkages (β-O-4, β-β, and β-5) were obtained by performing a sequential solvent extraction of kraft lignin (KL). Subsequently, spherical lignin nanoparticles (KLNPs, i.d. 193 ~ 1,039 nm) were synthesized by the nanoprecipitation process. The low-molecular-weight KL fractions generated KLNPs with a relatively enlarged diameter but less size-distributed. Further analysis showed that molecular weight, total, and phenolic hydroxyl group content in the lignin highly correlated with nanoparticle size. Every KLNPs exhibited comparable and good colloidal stability (-44.5 ~ -29.1 mV). Cell viability and hemotoxicity assessments revealed higher and sustained biocompatibility of KLNPs even at a high concentration, at least for three days. Second, KL was utilized as a polymer for the drug-encapsulated nanoparticle (ELNP) synthesis. Successful drug encapsulation of ELNPs by nanoprecipitation was determined (max. 59% for coumarin 6 and max. 73% for doxorubicin (DOX)). Compared to KLNP, the size (i.d. 176 ~ 469 nm) and surface charge (-40.5 ~ -32.7 mV) of ELNP showed no remarkable change regardless of introducing the drug in the particle. The drug release profile of ELNPs was faster in the relatively acidic condition (over 70% within 0 ~ 4 h) than the neutral (less than 50% within 0 ~ 4 h). In vitro cytotoxicity assay revealed the drug release effect of ELNPs, delaying but not decreasing the anticancer activity. In addition, the hemocompatibility test, in vivo allergic reaction test, and tumor inhibition assays using mice tumors showed high ELNP biocompatibility and side effect reduction during the chemotherapy. Lastly, alkaline/soda lignin (AL) was sequentially solvent-extracted, or its phenolic hydroxyl groups were chemoselectively methylated. These lignins with the modified structure were handled to form nanoparticles (ALNP) to clarify the effect of the lignin structure on the physical properties of nanoparticles. Pure ALNPs were obtained from neat lignin, solvent-extracted fractions (ALNP-Fs, i.d. 414 ~ 1,214 nm), and methylated lignins (ALNP-Ms, i.d. 516 ~ 721 nm). Specifically, the size properties of ALNP-Ms showed a very high negative correlation (R2 = 0.95) with the phenolic hydroxyl group amount. It indicates that the phenolic hydroxyl groups in lignin are the main structures that affect the nucleation and/or condensation during the nanoprecipitation process. ALNPs exhibited high colloidal stability (-42.3 ~ -32.7 mV), and most of them also showed good in vitro biocompatibility.최근 리그닌을 약물 전달 시스템의 재료로 이용하는 연구가 주목받고 있다. 특히 리그닌으로 나노입자를 제조하는 연구는 약물 수송 및 방출 조절에 관한 장점을 부여할 수 있어 선호되는 추세이다. 하지만 그중 리그닌의 구조적 특성이 나노입자의 형성 과정이나 물성(크기, 콜로이드 안정성, 생체안전성)에 미치는 영향에 주목한 연구는 거의 없는 실정이다. 본 연구에서는 산업공정 부산물로 발생하는 리그닌 부산물의 구조적 특성이 리그닌 나노입자의 물성에 미치는 영향을 이해하고자 하였다. 해당 결과를 바탕으로 약물 저장 리그닌 나노입자를 합성하여 약물 방출 특성과 기대 효과를 생체외 및 생체내 조건에서의 생체안전성 실험을 통하여 구명하였다. 우선 산업공정 부산물인 kraft 리그닌을 순차적 용매 분획화하여 분자량, 수산기 함량, 분자간 주요 결합 함량이 각기 다른 5개 분획으로 분리하였다. 각 리그닌 분획으로부터 THF-리그닌 용액과 증류수의 치환에 의한 나노침전법을 통하여 나노입자를 획득하였으며 입자 크기는 최소 193 nm에서 최대 1,039 nm로 다양하게 나타났다. 리그닌 분자량이 작을수록, 페놀성 수산기 함량이 높을수록 크면서 균질한 분포를 보이는 입자가 형성되었으며 kraft 리그닌의 구조적 특성과 형성된 나노입자의 입자 크기와 분포는 리그닌 분자량과 수산기 함량, 특히 페놀성 수산기 함량에 영향을 받는 것으로 판단되었다. 나노입자는 대체로 깔끔한 구형으로 형성되었으며 증류수에 안정적인 콜로이드 상태로 분포하였다(-44.5 ~ -29.1 mV). 세포 생존율 및 혈구독성 평가 결과 리그닌 나노입자는 높은 농도 조건에서도 최소 3일간 높은 수준의 지속적인 세포안전성을 보이는 것을 확인하였다. 위 결과를 바탕으로 선택한 특정 리그닌 조건으로부터 약물 저장 나노입자를 합성하였으며 약물 방출 조절 효과를 입증하기 위한 실험을 수행하였다. 약물 저장 리그닌 나노입자는 coumarin 6에 대하여 최대 59%, 독소루비신에 대하여 최대 73%의 약물 저장 효율을 나타내었다. 약물 저장 리그닌 나노입자의 크기(176 ~ 469 nm)는 대조군과 비교하였을 때 약물 첨가에 큰 영향을 받지 않는 것으로 나타났다. 또한, 약물 저장 여부는 나노입자의 콜로이드 안정성에도 영향을 미치지 않았다. 약물 저장 리그닌 나노입자는 대체로 0 ~ 4시간 사이에 빠른 약물 방출 경향을 보였고 혈액과 동일한 pH 조건보다는 산성 조건에서의 약물 방출이 더 빠른 것으로 나타났다. 리그닌 나노입자는 생체외 혈구독성 실험과 생체내 알레르기 반응 실험에서 우수한 생체안전성을 나타냈으며, 생체외 세포독성 실험과 생체내 항암효과 검증 실험에서 약물 방출 조절 및 부작용 경감에 의한 사망률 감소 효과를 나타냈다. Alkaline/soda 리그닌의 경우 순차적 용매 분획화와 별도로 페놀성 수산기의 메틸화 반응을 수행하여 분자량의 변화가 상대적으로 적으면서 페놀성 수산기의 함량이 각기 다른 4개 그룹을 추가로 획득하였다. 구조적 변형을 거친 해당 리그닌 그룹으로부터 나노입자를 획득하여 그 물성을 분석한 결과, 분획화 리그닌 유래 나노입자는 kraft 리그닌에서와 유사한 크기 분포 경향을 보였다. 반면, 메틸화 리그닌 유래 나노입자는 페놀성 수산기가 감소함에 따라 크기가 감소하는(721 ~ 516 nm) 뚜렷한 경향을 보였다. 모든 조건에서 나노입자의 콜로이드 안정성은 높게 유지되었으며 생체안전성 역시 대부분 농도 및 시간 조건에서 안전성을 나타냈다. 본 연구에서는 펄프 및 바이오에탄올 생산공정에서 부산물로 발생하는 리그닌의 고부가가치 활용방안으로 약물 방출 조절 나노입자 합성을 제시하였다. 리그닌의 구조적 특성과 나노입자 물성의 상관성을 구명하여 본 연구에서 다루지 않은 다른 리그닌을 이용한 나노입자 제조 및 물성 제어에 있어서도 단초를 제공하였으며, 생체외 및 동물 실험을 통한 리그닌 나노입자의 약물 방출 조절 및 부작용 경감 효과를 검증하여 약물 전달체 원료로써 실제 적용 가능성을 제시하였다.Contents Chapter 1 Introduction 1 1. Background 2 1.1. The concept of lignocellulosic biorefinery 2 1.2. Lignin production from pulping and sugar-based biorefinery 3 1.3. Understanding the lignin structure for further application 6 1.4. Polymeric nanoparticle for biomedical use 8 2. Objectives 12 3. Literature review 15 3.1. Valorization of lignin macromolecules 15 3.1.1. Isolation of lignin from biomass 15 3.1.2. Determination of lignin structure 19 3.1.3. Lignin fractionation 24 3.1.4. Structural functionalization of lignin 28 3.2. Nano-sized carriers for drug delivery 38 3.2.1. Strategies for drug delivery 38 3.2.2. Nanoparticle synthesis 44 3.2.3. Strategies for controlled drug release 48 Chapter 2 Synthesis of pure nanoparticles with solvent-fractionated kraft lignin and evaluation of their biocompatibility 51 1. Introduction 52 2. Materials and methods 54 2.1. Materials 54 2.2. Lignin fractionation 56 2.3. Characterization of fractionated lignin 58 2.4. Synthesis of lignin nanoparticle 62 2.4.1. Pre-dialysis concentration 62 2.4.2. Lignin fractions 64 2.5. Characterization of lignin nanoparticle 65 2.6. Biocompatibility tests 66 2.6.1. CCK-8 assay 66 2.6.2. Hemolysis assay 68 2.7. Statistical analysis 69 3. Results and discussion 70 3.1. Characteristics of lignin fractions 70 3.2. Particle size and morphology 80 3.3. Particle surface charge 89 3.4. Storage stability of the particle in pure water 90 3.5. In vitro cell viability 93 3.6. Hemocompatibility 98 4. Conclusion 100 Chapter 3 In vivo and in vitro evaluation of drug-encapsulated lignin nanoparticle 101 1. Introduction 102 2. Materials and methods 106 2.1. Materials 106 2.2. Animals and ethics 108 2.3. Synthesis of encapsulated lignin nanoparticle 109 2.3.1. Coumarin 6-encapsulated nanoparticle 109 2.3.2. DOX-encapsulated nanoparticle 110 2.4. Characterization of encapsulated nanoparticle 111 2.4.1. Determination of physical properties of nanoparticle 111 2.4.2. Drug loading 112 2.4.3. In vitro release study 113 2.5. Biocompatibility tests 114 2.5.1. CCK-8 assay 114 2.5.2. Hemolysis assay 115 2.5.3. In vivo detection of an allergic reaction 116 2.6. In vivo anticancer efficacy 117 2.7. Statistical analysis 118 3. Results and discussion 119 3.1. Particle size and encapsulation efficiency 119 3.1.1. Coumarin 6-encapsulated nanoparticle 119 3.1.2. DOX-encapsulated nanoparticle 124 3.2. Particle surface charge 128 3.3. In vitro drug release 130 3.4. In vitro cytotoxicity 135 3.5. Hemocompatibility 138 3.6. Hypersensitivity test 140 3.7. In vivo antitumor efficacy 142 4. Conclusion 145 Chapter 4 Effect of chemoselective methylation of the phenolic hydroxyl group on lignin nanoparticle synthesis 147 1. Introduction 148 2. Materials and methods 150 2.1. Materials 150 2.2. Functionalization of lignin 152 2.3. Characterization of lignin 154 2.4. Synthesis of lignin nanoparticle 156 2.4.1. Pre-dialysis concentration 156 2.4.2. Functionalized lignin 158 2.5. Characterization of lignin nanoparticle 159 2.6. Biocompatibility test 160 2.7. Statistical analysis 161 3. Results and discussion 162 3.1. Characteristics of functionalized lignin 162 3.1.1. Lignin fractions 162 3.1.2. Lignin methylation 174 3.2. Effect of lignin characteristics on nanoparticle size 178 3.2.1. Effect of solvent fractionation 178 3.2.2. Effect of the hydroxyl group 186 3.3. Particle surface charge 190 3.4. Storage stability of nanoparticle 192 3.5. In vitro cell viability 195 4. Conclusion 198 Chapter 5 Conclusion 199 References 203 초록 225Docto

Gelatin Nanoparticles as Potential Nanocarriers for Macromolecular Drugs

With an objective of designing gelatin based nanoparticulate delivery system for macromolecules, some of the important challenges associated with gelatin nanoparticles are addressed in this thesis. The first goal is to avoid aggregation, one of the most often encountered problems during nanoparticle formation from gelatin. In this context, different parameters involved in nanoprecipitation technique are investigated, in order to obtain optimum preparative conditions. Effective loading of FITC-dextran as a model hydrophilic macromolecule shows good potential of the system for macromolecular drugs. Attempts are also made to modify the surface of gelatin nanoparticles with PEI, in order to facilitate surface adsorption of negatively charged macromolecules. However, the size of nanoparticles is substantially increased after PEI coating. Moreover, the particles strongly positively charged particles showed an increased toxic behavior after surface modification with PEI. The second main challenge in gelatin nanoparticles is the use of crosslinkers for stabilization of particles. Since crosslinkers not only crosslink gelatin but also the active sites of therapeutic proteins, this may lead to biological inactivity of proteinaceous compounds. Therefore, we introduce an alternative approach of stabilization. Gelatin nanoparticles are entrapped in nanospheres made of synthetic polymers, using a unique technique of nanoprecipitation-emulsion solvent evaporation. PLGA seems to be ineffective, while Eudragit® E100 efficiently entraps gelatin nanoparticles in nanosphere matrix depending on concentration

Self-assembled hydrogel nanoparticles for drug delivery applications

Hydrogel nanoparticles—also referred to as polymeric nanogels or macromolecular micelles—are emerging as promising drug carriers for therapeutic applications. These nanostructures hold versatility and properties suitable for the delivery of bioactive molecules, namely of biopharmaceuticals. This article reviews the latest developments in the use of self-assembled polymeric nanogels for drug delivery applications, including small molecular weight drugs, proteins, peptides, oligosaccharides, vaccines and nucleic acids. The materials and techniques used in the development of self-assembling nanogels are also described

Chapter 11 – Polymeric micro- and nanoparticles for controlled and targeted drug delivery

Nanotechnology has great potential in the field of medicine and pharmacy because nano objects have comparable dimensions to biological entities. Polymer-based particles play an integral role as vehicles in the controlled delivery of different forms and types of active substances, such as anticancer drugs, antihypertensive and immunomodulatory agents, medical imaging contrast media, hormones, vitamins, and different macromolecules, such as nucleic acids (deoxyribonucleic acid, ribonucleic acid), proteins, antibodies, etc. The release of the active agent may be constant over a long period, it may be cyclic over a long period, or it may be triggered by the environment or other external events. The purpose behind controlling the drug delivery is to achieve more effective therapies while eliminating the potential for both under and overdosing. Other benefits of using controlled-delivery systems can include the maintenance of drug levels within a desired range, the need for fewer administrations, making optimal use of the drug in question, and increased patient compliance. This review article reports on obtaining polymeric micro- and nanoparticles with a special emphasis on obtaining polyester particles, the incorporation of different active substances within a polymer matrix, the degradation and release process of active substances from the polymeric particles, the physiochemical and biological properties of such obtained systems, as well as their application as drug-delivery systems.This is the accepted manuscript of the book chapter: Wu, T.-J., Chiu, H.-Y., Yu, J., Cautela, M.P., Sarmento, B., das Neves, J., Catala, C., Pazos-Perez, N., Guerrini, L., Alvarez-Puebla, R.A., Vranješ-Đurić, S., Ignjatović, N.L., 2018. Nanotechnologies for early diagnosis, in situ disease monitoring, and prevention, in: Nanotechnologies in Preventive and Regenerative Medicine. Elsevier, pp. 1–92. [https://doi.org/10.1016/B978-0-323-48063-5.00001-0

Surface Crosslinked Gelatin Nanoparticles As New Tool for the Delivery of Hydrophilic Macromolecular Drugs

For the delivery of hydrophilic macromolecules, a novel flexible, hydrophilic delivery system based on gelatin nanoparticles is developed. Stabilization of gelatin in water is addressed using an apolar zero length crosslinker, i.e., diisopropylcarbodiimide (DIC). Crosslinking of GNPs with polar crosslinkers has certain limitations not only crosslinking gelatin nanoparticles but also the loaded proteins, which interfere not only in the release of cargo but also their biological activity. Therefore, we introduced a novel crosslinking approach termed as interfacial crosslinking with the application of DIC which specifically crosslink the colloidal interface and not diffusing into the interior of nanoparticle. In this context, critical process parameters involved in the crosslinking process have been investigated in order to obtain optimum preparation conditions. The main challenge while formulating these surface crosslinked gelatin nanoparticles (scGNPs) was purification. The centrifugation results in the formation of non redispersible pellet. Therefore, we optimized tangential flow filtration as a promising alternative purification tool. For the final proof of our hypothesis that the hydrophobic crosslinker crosslink only the colloidal interface of GNPs without crosslinking the loaded protein, the surface crosslinked GNPs were loaded with a model hydrophilic protein, i.e., lysozyme. This proved scGNPs as a flexible delivery system for protein based drugs

MICROENCAPSULATION OF FEED ADDITIVES WITH POTENTIAL IN LIVESTOCK AND POULTRY PRODUCTION: A SYSTEMATIC REVIEW

The expected population growth will increase global food consumption, particularly meat consumption, which is estimated to increase 14% by 2030. Hence, the efficient utilization of all the resources involved in meat production, predominantly feed additives in livestock, is important due to economic costs and the high environmental in terms of gas production and ammonia excretion. Efforts have been made to increase efficiency in livestock production and improve the absorption and utilization of nutrients. Nevertheless, advances in technology in the chemical, pharmaceutical, and food industries have barely been used by the livestock and poultry industry. The micro/nano encapsulation process has been used in animal nutrition to protect bioactive compounds or to control the release of feed additives into the animal gastrointestinal tract, avoiding rumen microbes attack, or monogastric digestion in swine and poultry, to be available in the small intestine. However, not all the encapsulation techniques are suitable for applications in animal feeding. For example, spray drying, emulsion and coacervation can be used to control the release of feed additives in ruminants. In this sense, micro encapsulation of different feed additives such as amino acids, fatty acids, and probiotics may face enormous challenges to help improve livestock and poultry nutrition. The objective of this review is to highlight and discuss the techniques, compounds, and key aspects involved in the encapsulation of feed additives and nutrients with potential applications in the livestock and poultry production

Natural-origin materials for tissue engineering and regenerative medicine

Recent advances in tissue engineering and regenerative medicine have shown that combining biomaterials, cells, and bioactive molecules are important to promote the regeneration of damaged tissues or as therapeutic systems. Natural origin polymers have been used as matrices in such applications due to their biocompatibility and biodegradability. This article provides an up-to-date review on the most promising natural biopolymers, focused on polysaccharides and proteins, their properties and applications. Membranes, micro/nanoparticles, scaffolds, and hydrogels as biomimetic strategies for tissue engineering and processing are described, along with the use of bioactive molecules and growth factors to improve tissue regeneration potential. Finally, current biomedical applications are also presented.The authors would like to thank to the financial support from the Portuguese Foundation for Science and Technology (FCT) for the fellowship grants of Simone S Silva (SFRH/BPD/112140/2015), Emanuel M Fernandes (SFRH/BPD/96197/2013), Joana-Silva Correira (SFRH/BPD/100590/2014), Sandra Pina (SFRH/BPD/108763/2015), Silvia Vieira (SFRH/BD/102710/2014), “Fundo Social Europeu”- FSE and “ Programa Diferencial de Potencial Humano POPH”, and to the distinction attributed to J.M. Oliveira under the Investigator FCT program (IF/00423/2012). It is also greatly acknowledged the funds provided by FCT through the project EPIDisc (UTAP-EXPL/BBBECT/0050/2014), financed in the Framework of the “International Collaboratory for Emerging Technologies, CoLab”, UT Austin|Portugal Program.info:eu-repo/semantics/publishedVersio